Impacts of Chronic and Cyclic Hypoxia on Gene Expression and Reproduction in Grass Shrimp

1. IMPACTS OF CHRONIC AND CYCLIC HYPOXIA ON GENE EXPRESSION AND REPRODUCTION IN GRASS SHRIMP, PALAEMONETES PUGIOMarius Brouwer; Nancy Brown-Peterson; Thea Brouwer; Steve Manning; Tiandao Li; Nancy Denslow R82945801 Department of Coastal Sciences, The University of Southern Mississippi, Department of Physiological Sciences , University of Florida 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 • 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 • Evaluate laboratory-derived parameters in field-collected grass shrimp

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4. Macroarray Membrane Layout Hsp70c 16kDa Hsp70 Hsp70 L5 PPI L13 S6 L31 L27 S2 L3 EF2 S20 L21 L6 CathC S14 CathL Apep Cprot Crus Apep Alip ACoA ACoA Vtg Chor Vtg CcOx Asyn CcOx cytb Asyn CcOx Amyl Hcy Hif Hcy Hcy Hcy Perl gbp pepck Chit SugT tub Cell myosin trop 16S rRNA actin haembp cuti odc fer cSOD mSOD mt1 hist odc Sp 2 Sp 3 Spike 3 Hybridized with RNA from 1.5 ppm DO 4 d grassshrimp

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. Gluconeogenesis Cyclic DO Phosphoenol Pyruvate Glucose GDP + CO2 AcylCoA dehydrogenase GTP PEP Carboxykinase fatty acid oxidation Cyclic DO TCA CYCLE

10. Chronic Hypoxia Day 14 Cyclic 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. Mobile Bay Sampling Sites Sampled 2004, 2005 Weeks Bay Mobile Bay EastBay Escambia Bay Pensacola Bay Sampling Sites Sampled 2002, 2003, 2004, 2005 Garcon Point EPA-GED

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 • Downregulation of mitochondrial gene transcription similar to changes induced by chronic 1.5 ppm DO exposures in the lab • Downregulation of hemocyanin transcription in late summer, not observed in lab exposures, but indicative of anaerobic metabolism • 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: EcologicallyImportant Parameters • chronic hypoxia • cyclic DO • Field

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

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

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

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. Reproduction of Grass Shrimp in the Field Weeks Bay, July 2004 Pensacola Bay, August 2004

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