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  • Not primarily reliant on visual or chemotaxis Figure: study showing groping vs pounce (webover) hunting being dependent on the environment
  • Chase and wells: chemicals being glutamic acid, AMP, and glycine Lee: chemicals being proline, ATP, and crab extract Problems with studies: Chase and Wells -> no specific direction set, used chemicals (not natural); Lee -> lab-reared animals that were selected for preferencing crab, used chemicals (not natural) My question: how would octopuses respond to a more natural trial of chemotaxis?
  • - By using live crabs
  • Explain tank and experimental set-up Essentially a Y-maze (no mixing between arms), 2 set-ups: blind and visual – blind trials = black plexi dividers so octopus couldn’t see the crab with opaque holed boxes for crabs with water flow through them, visual = clear plexi dividers with plexi boxes for crabs
  • Used 2 octos: to try and reduce personality induced behavior Allowed for acclimation time, then removed grating and gave the octopus 40 min to make a choice A choice was made when the octopus’ head passed around the T structure into one arm of the maze Type of trial and the arm containing the prey were randomized to reduce learned behavior
  • Behavior: color changes/responses to visual clues
  • Mention briefly a little bit about the crab species: Green shore crab, local species, grows up to 5 cm, lives in tide pools, common SDS-PAGE for protein analysis, PCR for gene expression
  • Salad spinner: 2.5 min Water bath for 1 hour Collect and freeze water samples After hour, immediately kill and remove gills Fixate one gill from each in Tri-Reagent for RNA isolation Fixate one gill from each in CelLytic to fixate protein
  • Hemocyanin: used to carry oxygen (of course there’d be a TON in the gills) Arginine kinase: helps maintain ATP levels by the phosphorylation of "phosphagens"  which serves as a high energy source from which ATP can be replaced quickly, and can bind actin Superoxide dismustase: destroys radical oxidative species (in mitochondria), which can be excessively generated during times of stress Brachyurin: serine protease that degrades collagen Cuticle protein: from exoskeleton (contamination)
  • Fabrizio_capstone

    1. 1. Anna Fabrizio 3/9/10 University of Washington School of Aquatic and Fisheries Sciences Chemotaxis and foraging behavior in Octopus rubescens
    2. 2. Octopus physiology <ul><li>octopuses are known to have well developed vision, often used for hunting </li></ul><ul><li>many octopuses live in habitats where vision is compromised </li></ul><ul><li>many octopuses prey on organisms that don’t move </li></ul><ul><li>octopus suckers are used for sensing “taste” </li></ul><ul><ul><li>most sensitive at the tips of arms </li></ul></ul>
    3. 3. Octopus foraging behaviors <ul><li>2 primary foraging behaviors: </li></ul><ul><ul><li>Poke/grope </li></ul></ul><ul><ul><li>Web-over </li></ul></ul><ul><li>Both are primarily tactile in nature, but dependent on visual range </li></ul>(Forsythe and Hanlon, 1995) Web-over Web-over Web-over Web-over
    4. 4. Octopus chemotaxis <ul><li>little research done on distance chemotaxis in octopuses </li></ul><ul><li>Chase and Wells (1986) </li></ul><ul><ul><li>Blind octopuses responded to and were able to sense the general direction of chemicals in the water </li></ul></ul><ul><li>Lee (1992) </li></ul><ul><ul><li>Octopuses in Y-maze responded to specific chemical direction </li></ul></ul><ul><li>research has been done on tactile/taste discrimination by octopuses (Wells 1962) </li></ul>
    5. 5. Objective <ul><li>to expand on previous chemotaxis studies by analyzing octopus reaction to live prey </li></ul><ul><ul><li>Octopus rubescens has not been used for chemotaxis studies in the past </li></ul></ul>
    6. 6. Behavioral experiments Experimental tank set-up T-wall boxes barrier wall
    7. 7. Behavioral experiments
    8. 8. Results <ul><li>19 trials performed: 8 blind, 11 visual </li></ul><ul><li>1 blind trial resulted in a choice (crab) </li></ul><ul><li>6 visual trials resulted in a choice </li></ul><ul><ul><li>all chose the crab </li></ul></ul><ul><li>Behavior observations </li></ul><ul><ul><li>more response to crabs during visual trials </li></ul></ul><ul><ul><ul><li>color change, active motion </li></ul></ul></ul>
    9. 9. What does this mean? <ul><li>Can we confidently determine that octopuses CAN use distance chemotaxis from previous studies done with chemicals alone? </li></ul><ul><ul><li>Natural foraging behaviors show little evidence for the use of distance chemotaxis </li></ul></ul><ul><ul><ul><li>But what about circumstances that compromise vision? </li></ul></ul></ul>
    10. 10. Stressed out crabs <ul><li>During trials, crabs also exhibited different kinds of behavior </li></ul><ul><ul><li>when crabs could see the octopus, they held very still but were obviously in distress </li></ul></ul><ul><li>Do octopuses respond more frequently to stressed crabs than non-stressed crabs? </li></ul><ul><ul><li>Is there a physiological difference between stressed/non-stressed crabs? </li></ul></ul>
    11. 11. Prey analysis – Hemigrapsus oregonensis <ul><li>Purpose: analyze shore crab RNA/protein to see if they express something that octopuses would respond to </li></ul><ul><li>Techniques: </li></ul><ul><ul><li>SDS/PAGE </li></ul></ul><ul><ul><li>PCR </li></ul></ul><ul><li>Specifically looking at stressed vs non-stressed </li></ul><ul><ul><li>stressed, immersed crabs excrete excessive ammonia, waste, carbon dioxide </li></ul></ul>
    12. 12. Procedure
    13. 13. Protein analysis <ul><li>protein fixation and extraction from gill tissues </li></ul><ul><li>SDS-PAGE gel </li></ul>C2 C3 C4 C5 C6 S1 S2 S3 S4 S5 S6
    14. 14. Results Genetic analysis <ul><li>RNA fixation and extraction from gill tissues </li></ul><ul><li>reverse transcription -> cDNA </li></ul><ul><li>4 primers, 3 genes – Carcinus maenas : </li></ul><ul><ul><li>CHH and CHH precursor 6: hormone expressed under stress </li></ul></ul><ul><ul><li>Metallothionein: cysteine-rich protein that helps reduce oxidative stress </li></ul></ul><ul><ul><li>Na+/K+ - ATPase: enzyme that helps regulate ammonia excretion </li></ul></ul>
    15. 15. Genetic analysis
    16. 16. <ul><li>further study into the significance of stress on prey and the ability of octopuses to find them </li></ul><ul><li>more extensive research on distance chemotaxis with live prey </li></ul>Future possibilities
    17. 17. Acknowledgments Steven Roberts, Sam White, Mackenzie Gavery Tim Carpenter, Kathryn Kegel - Roberts lab - Seattle Aquarium
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