1. Response of the Pacific Oyster Crassostrea gigas to multiple stressors: An integrated molecular approach INTEGRATIVE ENVIRONMENTAL PHYSIOLOGY David Berman SammiBrombacker Dave Metzger Paul Pratt Jason Tayag Ross Whippo 15 DECEMBER 2010

4. General Methods

5. Experimental Design

6. Specific Methods and Results

7. What does this all mean?D. Berman

9. May be ‘natural’ or anthropogenically induced

10. Stressors act synergistically

11. Organisms use a variety biological mechanisms to copehttp://www.paranormalknowledge.com/wp-content/uploads/2010/01/water-pollution.jpg http://www.solutions4pollution.org/pollution.jpg http://www.onlinefast.org/gaia/sites/default/files/photos/shannon%20point%20-%20algae%20bloom.jpg

15. Integrative Approach RNA Protein DNA Physiological Response R. Whippo http://commons.wikimedia.org/wiki/File:Western_blot.jpg

17. Valuable shellfish industry

18. Intertidal and Subtidal

19. Model organism as bioindicator

20. Sessile

22. Easy access

23. Cobble

24. n = 32S. Roberts http://winksite.mobi/xhtml/ms_fo_pg_v.cfm?fid=15611&id=13825&susid=25641&s=1&s2=1

27. Ubiquitous aquatic contaminant

28. Immunosuppressive effects

29. Bioaccumulates in oyster tissues

30. CuSO4 at 130ppmhttp://www.writeonnewjersey.com/tag/copper-penny/

31. Experimental Design 11/11/10 Collect Oysters from Big Beef Creek 11/12/10 OYSTERS ACCLIMATED (4 DAYS) n = 32 No treatment n = 16 Copper treatment n = 16 11/16/10 Vibrio treatment n = 8 Vibrio treatment n = 8 11/18/10 http://www.eol.org/pages/97174

33. Reemerging bacterial pathogen of shellfish

34. Linked to massive oyster mortality events

35. Correlated with increased sea surface temperature

36. Concentration 103 CFU/ml11/12/10 OYSTERS ACCLIMATED (4 DAYS) n = 32 No treatment n = 16 Copper treatment n = 16 11/16/10 Vibrio treatment n = 8 Vibrio treatment n = 8 11/18/10 http://www.eol.org/pages/97174

37. Experimental Design 11/11/10 Collect Oysters from Big Beef Creek 11/12/10 OYSTERS ACCLIMATED (4 DAYS) n = 32 No treatment n = 16 Copper treatment n = 16 11/16/10 Vibrio treatment n = 8 Vibrio treatment n = 8 11/18/10 11/19/20 SAMPLE n = 32

40. Disease causing agent in cholera (V. cholerae), paralytic shellfish poisoning (V. parahaemolyticus), and more!

41. Foodborne infections from eating undercooked seafood.V. Vulnificus CDC public health library

44. Physiological effects of Cu(II) on Vt Measured mRNA expression of RseA. What’s RseA? - Negatively regulates activity of RpoE

45. RpoE Extracellular stress response (thermal and chemical). Induced in response to protein misfolding. Induced upon Cu(II) exposure in E. coli.

46. RpoE and Copper Influence of 2mM Copper on growth of rpoE mutants in E. coli Wild-type rpoE deletion rpoE deletion complemented in trans Egler, M., Grosse, C., Grass, G., Nies, D. 2005. Role of the extracytoplasmic function protein family sigma factor RpoE in metal resistance of Escherichia coli. J. Bacteriol. 187(7). 2297-2307.

47. RpoE transcriptional regulation RseA negative regulated rpoE function Missiakas, D., Mayer, M.P., Lemaire, M., Georgopoulos C., Raina, S. (1997). Modulation of the Escherichia coli sE (RpoE) heat-shock transcription-factor activity by the RseA, RseB and RseC proteins. Mol. Micro. 24(2), 355-371.

48. Methods 500ml 2um filter

52. What can it tell us?

53. Methylation in oysters

54. Sample processing

55. What did I find?http://commons.wikimedia.org/wiki/File:DNA-fragment-3D-vdW.png

57. Found in vertebrates and invertebrates

58. Influences expression of genes

59. Normal part of organismal physiology - Epigenetics

60. Heritable!GATAGATACGATAGATA DNA UNMETHYLATED methyl group GATAGATACGATAGATA DNA METHYLATED

62. Shown to have ‘mosaic methylation’

63. Housekeeping genes

66. From mantle tissue

67. Nine individuals, three each from control, copper, vibrio

68. Enzyme digestions (CCGG)HpaII MspI UNMETHYLATED UNMETHYLATED METHYLATED METHYLATED

70. From mantle tissue

71. Nine individuals, three each from control, copper, vibrio

72. Enzyme digestions (CCGG)HpaII MspI CUT CUT UNMETHYLATED UNMETHYLATED CUT METHYLATED METHYLATED

74. Hsp70 selected as gene of interest

75. Regularly methylated

77. Critical threshold set manually

80. Copper sample did not amplify properly

81. Extractions

83. No clear pattern associated with stressors

84. Need to look deeper into methylation patterns and stress

85. How often is Hsp70 unmethylated?

88. Protect proteins from unfolding

89. Mark denatured proteins

90. Stress inducible

91. Associated with a cellular response when exposed to heavy metals (Boutet et al., 2003 and Boutet et al., 2004).photo: SGC

92. RNA Methods RNA was extracted from Big Beef Creek C. gigasgill tissue using TRI Reagent® Quantified using Nanodrop spectrophotometer 50 μl of each RNA sample was DNased using Turbo DNA-free™ Kit RNA templates were normalized to 2 μg/RT reaction All samples were reverse transcribed 225 uL of nuclease free water was added for a final dilution photo: bioweb.wku.edu

94. HSP 70

95. Copper oxidase - David

96. Metallothionein IV - Jason

97. 18s Ribosomal subunit was used as a reference

98. qPCR reaction ingredients were 1x Immunomix master mix, 500 nM forward and reverse primers, and 0.2 mM Syto-13 dye

99. 2 μlcDNA = template

101. A Cu sample had a low melt temperature

102. Vt in the absence of Cu lowers expression, but in the presence almost eliminates

104. Demonstrate a decrease in expression (Moraga et al., 2005)

105. Demonstrate an increase in expression in Mytilusedulis (Sanders, 1988)

106. Metals (especially copper) have an inhibitory effect on the HSP protein synthesis after 7 days (Moraga et al., 2005)

107. Using tissue exposed to the environment shows the most success

108. gill

110. Metallothioneins (MT) Thiol (cysteine)-rich Regulate useful metals in diet (Zn) Bind heavy metals (Cu, Hg, Cd, etc.) and transports out of cells May be affected by bacteria (Paul-Pont et al. 2010) MT-IV should increase transcription for gene with an increase in Cu2+

111. MT structure varies

112. Metallothionein IV Results qPCR amplification Vibrio tubiashii really seems to inhibit MT-IV expression Anova P=0.09 – More likely the result of too few replicates. Vt means were really different.

113. Figure 1: Average MT-IV expression

114. Metallothionein IV Decrease in total MT protein with copper exposure (Geretet al. 2002) Being used up and cannot be replenished because transcription of the gene is inhibited. Oxidative stress uncontrolled Oxidative stress leads to cancer, death Cu accumulates, harmful for consumption

115. Copper Oxidase

116. What does it do? Is a defense mechanism of Oysters and many other organisms against copper which is extremely toxic (Grass 2001) Binds copper to itself to keep it from causing harm to the cells by interrupting cell processes Oysters can store this copper in their tissues In high concentration turns their tissue green

117. Methods RNA was extracted and then Dnased to get rid of any carryover DNA Reverse Transcription to change RNA into cDNA Used qPCR amplification to test for gene expression

118. Copper Oxidase Expression in Oysters

119. Detrimental Effects of Vibrio

120. Things to Consider This data shows that Vibrio is inhibiting the transcription of copper-oxidase in some way This is extremely detrimental and leaves the oysters subject to copper poisoning Shows the importance of multiple stressor testing by showing clearly how one stressor can leave an organism defenseless to another

121. Western Blot

122. Methods Protein Extraction SDS-PAGE Gill Tissue Extraction http://ycl.enicp.net/web/ycl/files/MiniVerticalElectrophoresisSystemEV3.jpg

123. Western Blot

124. Image Analysis

125. Data & Analysis

126. Questions?