1. 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)

2. Objectives Grass Shrimp Hypoxia Project Clone hypoxia (chronic and intermittent)-responsive genes using subtractive hybridization and use clones to construct macroarrays Clone hypoxia (chronic and intermittent)-responsive genes using subtractive hybridization and use clones to construct macroarrays Use the gene arrays to identify hypoxia-responsive genes in grass shrimp in controlled laboratory experiments Use the gene arrays to identify hypoxia-responsive genes in grass shrimp in controlled laboratory experiments Identify hypoxia-responsive reproductive parameters in laboratory experiments – needed to scale up to the population level Identify hypoxia-responsive reproductive parameters in laboratory experiments – needed to scale up to the population level Evaluate laboratory-derived parameters in field- collected grass shrimp Evaluate laboratory-derived parameters in field- collected grass shrimp

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4. 2345678910 B C D E F G H I J K L HifHcy pepck actin Hsp70c 16kDaHsp70 PPI Hsp70 L13 L5 S6L31L27S2 L21 L3EF2 L6 S20 S14CathLApep CathC CprotCrusApep ACoA Vtg Alip ChorVtg CcOx cytbAsynCcOx AsynCcOx Amyl PerlgbpChitSugT tub16S rRNA trop haembp fercSOD Sp 2 Sp 3Spike 3 hist cutiodc mt1 Hybridized with RNA from 1.5 ppm DO 4 d grass shrimp Cell myosin mSOD Macroarray Membrane Layout

5. Gene Expression Studies Chronic hypoxia - laboratory Cyclic Hypoxia - laboratory Cyclic hypoxia - field studies

7. Chronic Hypoxia Day 3 Cyclic Hypoxia Day 3

8. Chronic Hypoxia Day 7 Cyclic Hypoxia Day 7

9. AcylCoA dehydrogenase PEP Carboxykinase TCA CYCLE Phosphoenol Pyruvate Glucose Cyclic DO Gluconeogenesis fatty acid oxidation Cyclic DO GTP GDP + CO 2

10. Cyclic Hypoxia Day 14 Chronic Hypoxia Day 14

11. 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

12. 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

13. 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

14. Gene expression profiles of grass shrimp form normoxic and hypoxic field sites

15. Weeks Bay Mobile Bay East Bay Garcon Point EPA- GED EscambiaBay Mobile Bay Sampling Sites Sampled 2004, 2005 Pensacola Bay Sampling Sites Sampled 2002, 2003, 2004, 2005

17. Gene Expression Relative to Normoxic Controls Similar across years and sites for cyclic hypoxia!

18. Seasonal Change/Recovery in Gene Expression

19. 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

20. 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

21. Reproductive Studies: Ecologically Important Parameters chronic hypoxia cyclic DO Field

22. 2-3 ppm DO 1.5 ppm DO P = 0.003 P = 0.027 P = 0.010 HHNNHN 0 200 400 600 800 1000 1200 A B B Relative Fecundity Hypoxia Increases Relative Fecundity

24. Hypoxia (2.5 ppm DO) Increases Survival of Starved Larvae from Hypoxic Females Brood 1Brood 2 4.0 4.5 5.0 5.5 6.0 6.5 7.0 Hypoxic Normoxic A A B B Larval Survival (days)

25. Effects of Cyclic Hypoxia (1.5 8 ppm DO) on Grass Shrimp Reproduction: Laboratory Experiments HHHNNN Days to Egg Production 0 10 20 30 HHHNNN 0 5 10 15 20 25 Treatment HHHNNN 0 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 HHHNNN Relative Fecundity (# eggs/g) 0 100 200 300 400 500 600 Treatment HHHNNN 0 100 200 300 400 500 600 First Brood Second Brood A A,B B Relative Fecundity in Cyclic Hypoxia

26. Cyclic DO decreases estimated population growth rate (in an aquarium)

27. 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

28. Station % Egged Ratio M:F WC18.2%1:1 WBM44.9%1.3:1 Weeks Bay, July 2004 Station % Egged Ratio M:F GP175.8%1.1:1 MC45.8%1.5:1 MP29.5%1.25:1 Pensacola Bay, August 2004 Reproduction of Grass Shrimp in the Field

29. 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

30. Thanks to students and staff and NOAA and EPA

33. Develop Microarrays with ~ 5000 Genes from Sheepshead Minnow Embryos and Larvae Responsive to Environmental Stressors  Chronic hypoxia – stratified systems  Intermittent hypoxia – tidal creeks  Polycyclic Aromatic Hydrocarbons  Endocrine disruptors  Toxic metals  Pesticides and herbicides  Effects of hypoxia and anthropogenic stressors on embryos/larvae development and gene expression