JUVENILES FROMAQUACULTURE

RESTOCKING OFNATURAL HABITATS

SPORTFISHING

COMMERCIALAQUACULTURE

COMMERCIALFISHING

AQUARIUMTRADE

ENVIROMENTALPOLLUTION

ASSESSMENT

HANDYCRAFTS &

TROPHEES

BYPRODUCTSUTILIZATION

DIGESTIVEENZYMES

• Native species from the Native species from the Atlantic coast of North Atlantic coast of North AmericaAmerica

• Availability of individuals Availability of individuals raised under controlled raised under controlled conditionsconditions

• Able to tolerate different Able to tolerate different physicochemical physicochemical conditions (e.g.saltwater, conditions (e.g.saltwater, OO22, , ammonia, etc) ammonia, etc)

• Top position in the food Top position in the food web and occasional web and occasional scavengerscavenger

• High metabolic rate (e.g. High metabolic rate (e.g. growth)growth)

• Continuous contact with Continuous contact with the bottomthe bottom

• Ancient fish (simple Ancient fish (simple metabolic pathways)metabolic pathways)

Establishment of lab and field

methodologies to asses the impact of

aquatic pollution by means of

biomarkers in Alligator gar (A. spatula)

Establishment of lab and field

methodologies to asses the impact of

aquatic pollution by means of

biomarkers in Alligator gar (A. spatula)

1) BIOMARKERS SELECTION:

Implementing lab techniques for the evaluation of different biomarkers

Determining the response of alligator gar, of different ages to known environmental disruptors

1) BIOMARKERS SELECTION:

Implementing lab techniques for the evaluation of different biomarkers

Determining the response of alligator gar, of different ages to known environmental disruptors

1) Esterases • Carboxylesterases• Acetyl cholinesterase• Butiryl cholinesterase

2) Detoxification Glutathione S-transferase

3) Oxidative stress Superoxide Dismutase

4) Tissue injury Alkaline phosphatase

1) Esterases • Carboxylesterases• Acetyl cholinesterase• Butiryl cholinesterase

2) Detoxification Glutathione S-transferase

3) Oxidative stress Superoxide Dismutase

4) Tissue injury Alkaline phosphatase

1) DetoxificationCytochrome P450(CYP1A)

2) Endocrine Disruption Vitellogenin

(VTG)

1) DetoxificationCytochrome P450(CYP1A)

2) Endocrine Disruption Vitellogenin

(VTG)

1) Hydrocarbure (H)-naphtophlavone

2) Pesticide (P) Organophosphorate (Diazinon) 3) Endocrine Disruptor (ED) 17-Estradiol 4) Control (C) Fish Oil (Menhaden Oil)

1) Hydrocarbure (H)-naphtophlavone

2) Pesticide (P) Organophosphorate (Diazinon) 3) Endocrine Disruptor (ED) 17-Estradiol 4) Control (C) Fish Oil (Menhaden Oil)

Alligator gar: 3/Treatments

1 year: 60 g; 23 cm 2 years: 309 g; 38 cm Treatments application 50 mg/1000g in fish oil Intraperitoneal injection (exposure: 48 h)Tissue samples: Liver (L); Plasma (P); Gills (G); Mucus (M)

Alligator gar: 3/Treatments

1 year: 60 g; 23 cm 2 years: 309 g; 38 cm Treatments application 50 mg/1000g in fish oil Intraperitoneal injection (exposure: 48 h)Tissue samples: Liver (L); Plasma (P); Gills (G); Mucus (M)

ALLIGATOR GARALLIGATOR GAR

500 L Fiber glass tanks

500 L Fiber glass tanks

Treatment application(50 mg / 1000 g)

Treatment application(50 mg / 1000 g)

FILTER

FILTER

TissuesampleTissuesample

Sample inliquid N2

Sample inliquid N2

BiomarkerDetermination

BiomarkerDetermination

•A group of isozymes mainly located in RERA group of isozymes mainly located in RER

•Involved in metabolism of a wide variety of xenobiotics Involved in metabolism of a wide variety of xenobiotics containing esther, thioesther and amide groups. containing esther, thioesther and amide groups.

•Highest affinity for pesticides than related family of Highest affinity for pesticides than related family of cholinesterases (acetyl and butiryl)cholinesterases (acetyl and butiryl)

•Pesticides cause activity inhibition in most tissues Pesticides cause activity inhibition in most tissues • e.g. Livere.g. Liver

•Other xenobiotics cause Increase of activityOther xenobiotics cause Increase of activity

ASSAY METHODASSAY METHODEnzyme kinetics in 96-well microtiter plate using Enzyme kinetics in 96-well microtiter plate using

p-nitrophenyl acetate as substrate (Munilla & Starck, 1990p-nitrophenyl acetate as substrate (Munilla & Starck, 1990)

0.2 ml0.2 mlsubstratesubstrate

0.2 ml0.2 mlsubstratesubstrate

0.02 ml0.02 mlBufferBuffer

0.020.02 mlmlEnzymeEnzymeextractextract

Plus

Abs.Abs.405 nm405 nm10 min.10 min.

Carboxylesterase activity in 1 year alligator gar

0

0.1

0.2

0.3

0.4

0.5

0.6

ED H C

Treatments

Abs

/min

/mg

prot

ein

L

G

A

B

B

A A A

Carboxylesterase activity in 2 years alligator gar

0

0.1

0.2

0.3

0.4

0.5

0.6

ED H C P

Treatments

Abs/

min

/mg

prot

ein

P

L

G

A A A A

A A A

B

ABA A B

•Nonspecific phosphomono-esterase that removes a Nonspecific phosphomono-esterase that removes a phosphate group and transfers it to other molecules.phosphate group and transfers it to other molecules.

•Pollutants cause activity variation in several tissues. Pollutants cause activity variation in several tissues.

•Increased activity in the plasma in a variety of disease Increased activity in the plasma in a variety of disease conditions.conditions.

•Biomarker of tissue injury.Biomarker of tissue injury.

ASSAY METHODEnzyme kinetics in 96-well microtiter plate using

4-nitrophenyl phosphate as substrate (Moyano et al , 1996)

0.2 ml0.2 mlsubstratesubstrate

0.2 ml0.2 mlsubstratesubstrate

0.02 ml0.02 mlBufferBuffer

0.020.02 mlmlEnzymeEnzymeextractextract

Plus

Abs.Abs.405 nm405 nm10 min.10 min.

Alkaline phosphatase activity in 1 year alligator gar

0

0.2

0.4

0.6

0.8

1

1.2

ED H C

Treatments

Abs

/min

/mg

prot

ein

L

G

A

B

B

A A A

Alkaline phosphatase activity in 2 years alligator gar

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

ED H C P

Treatments

Abs

/min

/mg

prot

ein

L

G

AB

B

B

A

A AA

A

•Vtg is an estrogen-inducible protein synthesized by the liver.

•Males do not produce Vtg in measurable quantities under normal conditions, however have the capacity to synthesize it.

•Many chemicals that are released into the environment possess estrogenic properties.

•Measurement of Vtg in males and juveniles provides an excellent biomarker for estrogenic compounds.

washing

VTG Alligator gar VTG

VTG VTG

VTG Saturation

defatted milk

Secondary antibody

washing

washing

washing

enzyme substrate OPD

VTG immobilization

VTG VTG

VTG

IncubationVTGStandar Vtg or Sample

anti-VTG

VTG+

Competition

VTGVTG VTG

VTG

incubation

VTG VTG

VTG

Optical density measure

VTG VTG

VTG

METHOD:METHOD:Homologus competitive Homologus competitive

ELISAELISA

(Mendoza et al., In Press)(Mendoza et al., In Press)

VTG concentration in 1 year Alligator gar

0102030405060708090

100

ED H C

Treatments

ng

de

vtg

/mg

pro

tein

L

G

M

A

B BAB B

A

VTG concentration in 2 years alligator gar

0

20

40

60

80

100

120

ED H C P

Treatments

ng

vtg

/mg

pro

tein

P

L

G

M

A

B B B

BiomarkerEnvironmental disruptorEnvironmental disruptor

HydrocarbureHydrocarbure PesticidePesticide EstrogenicEstrogenic

Carboxyl -Carboxyl -esteraseesterase

Slight Reduction

(1 year)Reduction Increase

Alkaline Alkaline phosphatasephosphatase

Slight Reduction Increase Increase

VitellogeninVitellogenin Increase