2. JUVENILES FROM AQUACULTURE JUVENILES FROM AQUACULTURE RESTOCKING OF NATURAL HABITATS RESTOCKING OF NATURAL HABITATS SPORT FISHING SPORT FISHING COMMERCIAL AQUACULTURE COMMERCIAL AQUACULTURE COMMERCIAL FISHING COMMERCIAL FISHING AQUARIUM TRADE AQUARIUM TRADE ENVIROMENTAL POLLUTION ASSESSMENT ENVIROMENTAL POLLUTION ASSESSMENT HANDYCRAFTS & TROPHEES HANDYCRAFTS & TROPHEES BYPRODUCTS UTILIZATION BYPRODUCTS UTILIZATION DIGESTIVE ENZYMES DIGESTIVE ENZYMES

3. Native species from the Atlantic coast of North AmericaNative species from the Atlantic coast of North America Availability of individuals raised under controlled conditionsAvailability of individuals raised under controlled conditions Able to tolerate different physicochemical conditions (e.g.saltwater, O 2, ammonia, etc)Able to tolerate different physicochemical conditions (e.g.saltwater, O 2, ammonia, etc) Top position in the food web and occasional scavengerTop position in the food web and occasional scavenger High metabolic rate (e.g. growth)High metabolic rate (e.g. growth) Continuous contact with the bottomContinuous contact with the bottom Ancient fish (simple metabolic pathways)Ancient fish (simple metabolic pathways)

4. Establishment of lab and field methodologies to asses the impact of aquatic pollution by means of biomarkers in Alligator gar (A. spatula)

5. 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

6. 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) Detoxification Cytochrome P450 (CYP1A) 2) Endocrine Disruption Vitellogenin (VTG) 1) Detoxification Cytochrome P450 (CYP1A) 2) Endocrine Disruption Vitellogenin (VTG)

7. 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)

8. ALLIGATOR GAR 500 L Fiber glass tanks 500 L Fiber glass tanks Treatment application (50 mg / 1000 g) Treatment application (50 mg / 1000 g) FILTERFILTER FILTERFILTER Tissue sample Tissue sample Sample in liquid N 2 Sample in liquid N 2 Biomarker Determination Biomarker Determination

9. A group of isozymes mainly located in RERA group of isozymes mainly located in RER Involved in metabolism of a wide variety of xenobiotics containing esther, thioesther and amide groups.Involved in metabolism of a wide variety of xenobiotics containing esther, thioesther and amide groups. Highest affinity for pesticides than related family of cholinesterases (acetyl and butiryl)Highest affinity for pesticides than related family of cholinesterases (acetyl and butiryl) Pesticides cause activity inhibition in most tissuesPesticides cause activity inhibition in most tissues e.g. Liver e.g. Liver Other xenobiotics cause Increase of activityOther xenobiotics cause Increase of activity

10. ASSAY METHOD Enzyme kinetics in 96-well microtiter plate using p-nitrophenyl acetate as substrate (Munilla & Starck, 1990 p-nitrophenyl acetate as substrate (Munilla & Starck, 1990) 0.2 ml substrate substrate 0.02 ml Buffer Enzymeextract Plus Abs. 405 nm 10 min.

13. Nonspecific phosphomono-esterase that removes a phosphate group and transfers it to other molecules.Nonspecific phosphomono-esterase that removes a 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 conditions.Increased activity in the plasma in a variety of disease conditions. Biomarker of tissue injury.Biomarker of tissue injury.

14. ASSAY METHOD Enzyme kinetics in 96-well microtiter plate using 4-nitrophenyl phosphate as substrate (Moyano et al, 1996) 0.2 ml substrate substrate 0.02 ml Buffer Enzymeextract Plus Abs. 405 nm 10 min.

17. 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.

18. washing VTG Alligator gar VTG VTG Saturation defatted milk Secondary antibody washing enzyme substrate OPD VTG immobilization VTG Incubation VTG Standar Vtg or Sample anti-VTG VTG + Competition VTG incubation VTG Optical density measure VTG METHOD: Homologus competitive ELISA (Mendoza et al., In Press)

21. Biomarker Environmental disruptor HydrocarburePesticideEstrogenic Carboxyl - esterase Slight Reduction (1 year) ReductionIncrease Alkaline phosphatase Slight Reduction Increase Vitellogenin