This document summarizes research on how environmental stressors and changes impact the expression of genes in oysters. The summary is: [1] The research program examines how environmental stressors such as pathogens, carbon dioxide, mechanical stress, and anthropogenic activities affect the oyster stress response at the transcriptome, proteome, and epigenome levels. [2] Studies found that multiple environmental stressors can interact to reduce an oyster's ability to respond to stress, and that characterizing gene expression in natural oyster populations exposed to different human impacts provided insight into their biology and adaptation. [3] DNA methylation is an important mechanism in oysters for transcriptional control that can be impacted

1. changes in the environment and changes in
expression: insight from oysters
Steven Roberts
University of Washington
School of Aquatic and Fishery Sciences

7. research program overview
environmental
stressors
shellfish

8. research program overview
environmental
stressors
}
chemotaxis
binding
protease cells
oxidative burst
antimicrobial hemolymph shellfish
serum

9. research program overview
pathogens
environmental carbon dioxide
stressors microbes mechanical stress
microbes
microbes
stress response
transcriptome
proteome shellfish
epigenome*

10. rationale
comparative
biology

11. rationale
aquaculture

12. rationale
environmental
sciences

13. today
pathogens
environmental carbon dioxide
stressors mechanical stress
anthropogenic activities
stress response
transcriptome
proteome oysters
epigenome*

14. outline
pathogens
1. Hemocyte gene carbon dioxide
discovery mechanical stress
anthropogenic activities
2. Multiple stressors
3. Characterizing
natural populations

15. hemocyte (plated) cDNA library
Prior to washing After washing

16. ESTs
modified from Roberts et al 2009

17. vibrio exposure
Roberts et al 2009

18. interleukin 17
•cytokine
•large number of cytokines found in vertebrates are not
found in invertebrates
•interleukin 17 is not similar to other interleukins
•vertebrates- interleukin expressed in
activated memory T cells

19. interleukin 17
Roberts et al 2008

20. interleukin 17
Roberts et al 2008

21. interleukin 17
Roberts et al 2008

22. summary
•interleukin 17 could be an early cytokine present before
divergence of vertebrates and invertebrates
•expression analysis indicates it is a rapid response
signaling molecule
•complete signaling pathway and presence of other
molecules is not known in the oyster

23. outline
pathogens
1. Hemocyte gene carbon dioxide
discovery mechanical stress
anthropogenic activities
2. Multiple stressors

24. multiple stressors

25. ocean acidification
Sabine et al 2004

26. ocean acidification

27. ocean acidification

28. mechanical
control
control
mechanical
CO2
960 ppm
control
24 hours 5 min 40 min

29. mechanical stress

30. mechanical
control
control
mechanical
CO2
960 ppm
control
24 hours 5 min

31. microbial community
Horner-Devine

32. summary
•multiple environmental stressors could contribute to
increase stressor susceptibility
•data suggests stress response could be at capacity and
not able to properly respond to secondary stressor
•organisms will likely adapt to chronic changes
•not clear how global change will affect normal
physiological processes

33. outline
pathogens
1. Hemocyte gene carbon dioxide
discovery mechanical stress
anthropogenic activities
2. Multiple stressors
3. Characterizing
natural populations

34. urban, agriculture, water
fowl, marinas, seals
low population, low fecal
coliform

35. Transcriptomics
16 million
~40 bp
HQ reads
16 million
~40 bp
HQ reads

36. 32 million reads
v
17 million matched
Sigenae consensuses
29 thousand features
Upregulated features | min 10 unique hits & 2 fold increase
1329 1316
22 specific 25 specific

37. low population, low fecal urban, agriculture, water
coliform fowl, marinas, seals

38. low population, low fecal urban, agriculture, water
coliform fowl, marinas, seals

39. Upregulation (4-fold)
low population, low fecal
coliform
urban, agriculture, water
fowl, marinas, seals

40. RNAseq vs quantitative PCR

41. low population, qPCR
low fecal coliform
urban, agriculture,
water fowl,
marinas, seals
steroid 17-alpha-hydroxylase

42. x fold =
qPCR
complement C1q
steroid 17-alpha-hydroxylase serine protease inhibitor
TNF-related protein 4
2 fold 3 fold 4 fold
gonadotropin-releasing
calmodulin-like metalloproteinase inhibitor 3
hormone II receptor
specific
13 fold 3 fold

43. gene discovery

44. summary (biology)
•limited categorical differences
•selection and adaptation will play a significant role in
physiological response
•other normal physiological processes are occurring
(out of sync)

45. summary (technical)
•promising technology
•issues include; genome plasticity, multiple isoforms,
lack of genome
•analysis is not trivial

46. What is the functional role of DNA methylation in shellfish?
How do environmental conditions impact the epigenome?

47. background
global methylation
no methylation
(except CpG islands)
mosiac pattern - ~40-60% methylation

48. 5-methylcytosine and DNA repair
cytosine uracil
deamination
5-methylcytosine thymine
Over time..
loss of methylated
CpGs

49. oysters
in
silico
analysis
of
~30k
gene
clusters

50. biological process
• DNA methylation is an important transcriptional control mechanism

51. environmental effects
sites

52. epigenetics: implications and direction
•Epigenetic variation will redefine our concept of
“genetic” diversity
•Novel mechanism impacted by environmental change
•long-term effects ?
•Candidate process that could explain other
phenomenon

53. conclusions & directions
• comparative and evolutionary aspects can provide valuable
insight into more complex systems
• with a better understanding of mechanisms, populations that
are better able to respond to stressors can be identified. This
information will be applicable for conservation, aquaculture,
and predicting ecosystem change.
• characterizing natural populations to better understand
biology and the environment will continue to be complex,
however deep sequencing will prove to be a valuable tool

54. acknowledgements
Yannick Gueguen (Ifremer)
Julien de Lorgeril (Ifremer)
Frederick Goetz (WATER Institute)
Giles Goetz (WATER Institute)
Samuel White (UW)
Rachel Thompson (UW - student)
Claire Horner-Devine (UW)
Mackenzie Gavery (UW - student) funding
Joth Davis (Taylor Shellfish) USDA-NRAC
Dustin Lennon (UW) NOAA SK Program
Paul Sampson (UW) UW-SAFS