Development of Biomarkers for Stress in Octopus Rachel Thompson School of Aquatic and Fishery Sciences May 15, 2009
Background <ul><li>Important part of local marine ecosystem </li></ul><ul><li>Popular attraction at zoos and aquariums </l...
Objectives <ul><li>Develop non-invasive sampling techniques for octopus </li></ul><ul><li>2.  Identify biomarkers of stres...
Non-Invasive Techniques <ul><li>Epidermal mucus collected using 3 methods: </li></ul><ul><li>1. Underwater </li></ul><ul><...
Objectives <ul><li>Develop non-invasive sampling techniques for octopus </li></ul><ul><li>2.  Identify biomarkers of stres...
Protein Gel Electrophoresis <ul><li>Goals: </li></ul><ul><li>- Verify protein content </li></ul><ul><li>- Visualize protei...
Protein Identification <ul><li>Prominent protein bands excised  </li></ul><ul><li>Sequenced using mass spectroscopy </li><...
Mass Spectroscopy <ul><li>Bands digested into smaller peptides </li></ul><ul><li>Mass of peptides determined, compared to ...
Proteins Isolated from Mucus <ul><li>Casein : Antioxidant peptide </li></ul><ul><li>  -Radical scavenging activity </li></...
Objectives <ul><li>Develop non-invasive sampling techniques for octopus </li></ul><ul><li>2.  Identify biomarkers of stres...
Heat Shock Proteins <ul><li>Class of proteins </li></ul><ul><li>High production during stressful conditions: </li></ul><ul...
Western Blot - HSP 70 <ul><li>Target HSP 70 using antibody: </li></ul><ul><li>Not detected in mucus </li></ul><ul><li>Smal...
Objectives <ul><li>Develop non-invasive sampling techniques for octopus </li></ul><ul><li>2.  Identify biomarkers of stres...
Behavior Monitoring   <ul><li>“ Cephcam” time lapse videos </li></ul><ul><li>Online viewer surveys </li></ul><ul><li>Activ...
Behavior Observations <ul><li>Time lapse  video s analyzed at random time intervals </li></ul><ul><li>Baseline activity le...
Objectives <ul><li>Develop non-invasive sampling techniques for octopus </li></ul><ul><li>2.  Identify biomarkers of stres...
Egg Development Laid January, 2009 Refrain from imposing stressful conditions
Observation of Developmental Stages Early: 8 weeks
Late: 12 weeks - Appearance of  chromatophores - Movement within egg
 
Egg Development <ul><li>Opportunity to explore gene expression over time  </li></ul><ul><li>Egg samples collected weekly <...
Developmental Genes <ul><li>Orthodenticle-like protein (OTX) </li></ul><ul><ul><li>Involved in brain morphogenesis </li></...
Real-Time PCR (qPCR) <ul><li>Used to amplify/quantify targeted gene </li></ul><ul><li>Fluorescent dyes bind to double stra...
Orthodenticle-like protein (OTX) -Expressed early in development  -100,000 fold increase over time
Hedgehog (Hh) -Expressed later in development <100 fold increase over time
Results-Gene Expression <ul><li>Orthodenticle-like protein expressed early on in development (after ~1 month) </li></ul><u...
Behavior Observations Prior to Egg-Laying Post Egg-Laying
Summary <ul><li>Non-invasive methods successful to an extent </li></ul><ul><li>Possible protein biomarkers identified </li...
Applications and Future Work <ul><li>Assess physiological condition of octopuses in wild and in captivity </li></ul><ul><l...
Acknowledgements <ul><li>Dr .  Steven Roberts </li></ul><ul><li>Sam White </li></ul><ul><li>Dr. Greg Jensen </li></ul><ul>...
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Thompson_MGpresentation

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R. Thompson's Mary Gates Research Symposium Presentation

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Thompson_MGpresentation

  1. 1. Development of Biomarkers for Stress in Octopus Rachel Thompson School of Aquatic and Fishery Sciences May 15, 2009
  2. 2. Background <ul><li>Important part of local marine ecosystem </li></ul><ul><li>Popular attraction at zoos and aquariums </li></ul><ul><li>Population declines due to environmental stressors </li></ul><ul><li>Previous stress physiology work </li></ul>Giant Pacific Octopus ( E. dofleini ) Red Octopus ( O. rubescens )
  3. 3. Objectives <ul><li>Develop non-invasive sampling techniques for octopus </li></ul><ul><li>2. Identify biomarkers of stress </li></ul><ul><ul><li>a) Discovery-based protein analysis </li></ul></ul><ul><ul><li>b) Targeted protein analysis </li></ul></ul><ul><ul><li>c) Behavioral changes </li></ul></ul>
  4. 4. Non-Invasive Techniques <ul><li>Epidermal mucus collected using 3 methods: </li></ul><ul><li>1. Underwater </li></ul><ul><li>2. Skin </li></ul><ul><li>3. Sampling container </li></ul>
  5. 5. Objectives <ul><li>Develop non-invasive sampling techniques for octopus </li></ul><ul><li>2. Identify biomarkers of stress </li></ul><ul><ul><li>a) Discovery-based protein analysis </li></ul></ul><ul><ul><li>b) Targeted protein analysis </li></ul></ul><ul><ul><li>c) Behavioral changes </li></ul></ul>
  6. 6. Protein Gel Electrophoresis <ul><li>Goals: </li></ul><ul><li>- Verify protein content </li></ul><ul><li>- Visualize protein </li></ul><ul><li>Success varied with sampling technique </li></ul>Container Underwater Skin
  7. 7. Protein Identification <ul><li>Prominent protein bands excised </li></ul><ul><li>Sequenced using mass spectroscopy </li></ul>
  8. 8. Mass Spectroscopy <ul><li>Bands digested into smaller peptides </li></ul><ul><li>Mass of peptides determined, compared to known proteins </li></ul><ul><li>Sequences of proteins likely found in sample obtained </li></ul>Description Sequence (P02662) Alpha-S1-casein precursor YLGYLEQ (P02662) Alpha-S1-casein precursor EPMIGVNQELAYFYPELFR (P02808) Statherin precursor RIGRFGYGYGPYQPVPEQPLYPQPYQPQYQQYTF (P81605) Dermicidin precursor [Contains: Survival-promoting peptide; DCD-1] DAVEDLESVGK
  9. 9. Proteins Isolated from Mucus <ul><li>Casein : Antioxidant peptide </li></ul><ul><li> -Radical scavenging activity </li></ul><ul><li> -Inhibits growth of E.coli on skin </li></ul><ul><li>Dermicidin : Antimicrobial, limits skin infection by potential pathogens </li></ul>
  10. 10. Objectives <ul><li>Develop non-invasive sampling techniques for octopus </li></ul><ul><li>2. Identify biomarkers of stress </li></ul><ul><ul><li>a) Discovery-based protein analysis </li></ul></ul><ul><ul><li>b) Targeted protein analysis </li></ul></ul><ul><ul><li>c) Behavioral changes </li></ul></ul>
  11. 11. Heat Shock Proteins <ul><li>Class of proteins </li></ul><ul><li>High production during stressful conditions: </li></ul><ul><li>- Temperature </li></ul><ul><li>- Pathogens </li></ul><ul><li>- Hypoxia </li></ul><ul><li>Functions: </li></ul><ul><ul><li>- Chaperone: Membrane transport, maintain conformations of proteins </li></ul></ul><ul><ul><li>- Monitor: Transportation to proteasomes </li></ul></ul>
  12. 12. Western Blot - HSP 70 <ul><li>Target HSP 70 using antibody: </li></ul><ul><li>Not detected in mucus </li></ul><ul><li>Small leg tissue samples taken weekly </li></ul><ul><li>HSP 70 detectable </li></ul>
  13. 13. Objectives <ul><li>Develop non-invasive sampling techniques for octopus </li></ul><ul><li>2. Identify biomarkers of stress </li></ul><ul><ul><li>a) Discovery-based protein analysis </li></ul></ul><ul><ul><li>b) Targeted protein analysis </li></ul></ul><ul><ul><li>c) Behavioral changes </li></ul></ul>
  14. 14. Behavior Monitoring <ul><li>“ Cephcam” time lapse videos </li></ul><ul><li>Online viewer surveys </li></ul><ul><li>Activity categories </li></ul>Cephcam watch <ul><li>Check appropriate boxes </li></ul><ul><li>Lights on </li></ul><ul><li>Moving </li></ul><ul><li>Out of frame </li></ul><ul><li>Tenacles extended </li></ul><ul><li>On side of aquaria </li></ul><ul><li>On bottom </li></ul>
  15. 15. Behavior Observations <ul><li>Time lapse video s analyzed at random time intervals </li></ul><ul><li>Baseline activity level established </li></ul>
  16. 16. Objectives <ul><li>Develop non-invasive sampling techniques for octopus </li></ul><ul><li>2. Identify biomarkers of stress </li></ul><ul><li>a) Discovery based protein analysis </li></ul><ul><li>b) Targeted protein analysis </li></ul><ul><li>c) Behavioral changes </li></ul><ul><li>3. Investigate influence of environmental stress on biomarkers </li></ul>
  17. 17. Egg Development Laid January, 2009 Refrain from imposing stressful conditions
  18. 18. Observation of Developmental Stages Early: 8 weeks
  19. 19. Late: 12 weeks - Appearance of chromatophores - Movement within egg
  20. 21. Egg Development <ul><li>Opportunity to explore gene expression over time </li></ul><ul><li>Egg samples collected weekly </li></ul><ul><li>RNA extracted, cDNA made via reverse transcription </li></ul><ul><li>Designed primers for 2 developmental genes </li></ul>
  21. 22. Developmental Genes <ul><li>Orthodenticle-like protein (OTX) </li></ul><ul><ul><li>Involved in brain morphogenesis </li></ul></ul><ul><ul><li>Expressed in early stages of development </li></ul></ul><ul><li>Hedgehog (Hh) </li></ul><ul><ul><li>Mediate development, growth, patterning and morphogenesis </li></ul></ul><ul><ul><li>Muscle development in cuttlefish </li></ul></ul><ul><ul><li>Expressed early in development </li></ul></ul>
  22. 23. Real-Time PCR (qPCR) <ul><li>Used to amplify/quantify targeted gene </li></ul><ul><li>Fluorescent dyes bind to double stranded DNA product </li></ul><ul><li>Fluorescence can be measured to determine relative amounts of DNA sequences produced </li></ul>
  23. 24. Orthodenticle-like protein (OTX) -Expressed early in development -100,000 fold increase over time
  24. 25. Hedgehog (Hh) -Expressed later in development <100 fold increase over time
  25. 26. Results-Gene Expression <ul><li>Orthodenticle-like protein expressed early on in development (after ~1 month) </li></ul><ul><li>- Consistent with literature </li></ul><ul><li>Hedgehog protein expressed later in development (~2 months) </li></ul><ul><li>- Expressed throughout development </li></ul><ul><li>- Low levels of fluorescence may not have been detectable </li></ul>
  26. 27. Behavior Observations Prior to Egg-Laying Post Egg-Laying
  27. 28. Summary <ul><li>Non-invasive methods successful to an extent </li></ul><ul><li>Possible protein biomarkers identified </li></ul><ul><li>Changes in behavior related to senescence and parental care, similar methods could be used to evaluate stress </li></ul><ul><li>Opportunity to look at basic gene expression patterns in larval development </li></ul>
  28. 29. Applications and Future Work <ul><li>Assess physiological condition of octopuses in wild and in captivity </li></ul><ul><li>Test effectiveness of biomarkers in indicating stress (experiment) </li></ul><ul><li>Compare protein expression between individuals </li></ul>
  29. 30. Acknowledgements <ul><li>Dr . Steven Roberts </li></ul><ul><li>Sam White </li></ul><ul><li>Dr. Greg Jensen </li></ul><ul><li>Shawn Larson and Roland Anderson of the Seattle Aquarium </li></ul>

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