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Analysis of Genes Isolated
 from Plated Hemocytes
   of the Pacific Oyster,
      Crassostrea gigas
             Steven Roberts
      School of Aquatic and Fishery Sciences
            University of Washington

                Rick Goetz
          Great Lakes WATER Institute
            University of Wisconsin
Goal



       Better understand stress response
Immune System




Laura Canesi, Gabriella Gallo, Miriam Gavioli, and Carla Pruzzo
Bacteria–hemocyte Interactions and Phagocytosis in Marine
Bivalves. 2002. Microscopy Research And Technique 57:469–476
Specific Objectives


   • Identify genes expressed in oyster
     hemocytes
   • Characterize response of select
     molecules following Vibrio challenge
Outline

   • cDNA Library Description
   • Select Functional Categories
     •    Proteases and Protease Inhibitors

     •    Receptor and Associated Proteins

     •    Transcription/Translation/Cell Cycle
Plated hemocyte cDNA library


                         Incubate 24 hours
Pacific Oyster           Wash in sterile seawater
   C. gigas              Extract with Tri-Reagent




 + sterile
 saltwater
Plated hemocyte cDNA library
  Prior to washing   After washing
Extract from tissue
     ESTs
                          Make cDNA
  Gene - DNA                     (Complementary DNA =
                                                 Copy)


                       Clone into a “Vector”
Transcript - mRNA
                               = cDNA Library

                    Randomly sequence clones =
                    “Expressed Sequence Tags”
                             (ESTs)
C. gigas plated hemocytes




                            Roberts et al 2009
C. gigas plated hemocytes




                      modified from Roberts et al 2009
Markers &
more
Markers &
more
C. gigas Vibrio challenge




                            Roberts et al 2009
Proteases and Antiproteases
    Gene     Function
Proteases and Antiproteases
    Gene     Function
Proteases and Antiproteases
    Gene     Function
                        Inhibitors
Proteases and Antiproteases
    Gene     Function


                        Cathepsins
Proteases and Antiproteases
    Gene     Function


                        Cathepsins
Proteases and Antiproteases
    Gene     Function
                        C. virginica
                        Homologs
Proteases and Antiproteases
    Gene
                       C. virginica
                       Homologs
Receptors and other proteins
    Gene     Function
                        Prostaglandin E2
                        EP4 subtype
Prostaglandin
                               other ESTs present



    lipoxygenases
       production of
       leukotrienes,
          lipoxins,
  hydroperoxy fatty acids


                            15-hydroxyprostaglandin
                                     dehydrogenase
                                  PGE2 breakdown
Prostaglandin




                Mackenzie Gavery - Poster
Receptors and other proteins
    Gene     Function



                        Ectodysplasin A2
                           Receptor
Transcription / Translation / Cell Cycle
     Gene       Function


                            Erythroid
                            differentiation-
                            related factor
Transcription / Translation / Cell Cycle
     Gene       Function   High-mobility
                           group proteins
Transcription / Translation / Cell Cycle
                           High-mobility
                           group proteins
Transcription / Translation / Cell Cycle
                           High-mobility
                           group proteins
Conclusions

    • Several novel genes were identified from
      C. gigas hemocytes
    • Hemocyte signaling plays important role in
      immune response
    • Stress response is diverse and complex
    •
Conclusions

    • Several novel genes were identified from
      C. gigas hemocytes
    • Hemocyte signaling plays important role in
      immune response
    • Stress response is diverse and complex
    •
                                h/t

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