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Immune response and
disease tolerance in shellfish

              Steven Roberts
             University of Washington
       School of Aquatic and Fishery Sciences
flickr | Basenisa
Stress
•   Harmful anthropogenic compounds
•   Temperature

•   Hypoxia

•   Harmful organisms
      Bacteria,Viruses, Parasites
Sam White
Innate Immune System

• Present from birth
• “Non-specific” - acts on many pathogens
  and does not show specificity within a
  pathogen type
• Does not become more efficient on
  subsequent exposure to same pathogen
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
flickr | cpurrin1
Outline

• Genes expressed in oyster hemocytes
• Dermo disease and mechanical stress
• Disease resistance in oysters (and abalone)
• Clams and QPX
cDNA Library




               Rick Goetz
Interleukin-17
gene expression level
(fold increase over minimum)
Dermo disease and mechanical stress




Maps: Google - Oyster photos: G. Burreson
Dermo disease and mechanical stress




                          Gary Wikfors and Inke Sunila
Stress and Disease
Resistance ?
MVineyard                   flickr | cpurrin1




               Roxannna Smolowitz
                  Rick Karney
Another example




          Dale Leavitt, Marta Gomez-Chiarri
Markers ?
Glenn VanBlaricom, Carolyn Friedman
Abalone
Glenn VanBlaricom, Carolyn Friedman
Carolyn Friedman
Mining ESTs
                         from similar species
Gene                    Molecular Function         Biological Process       Cellular Component

rab1                    GTPase activity            vesicle-mediated         golgi apparatus
                                                   transport
rab7 (WSSV receptor) GTPase activity, protein      intracellular protein    golgi apparatus,
                         binding                   transport                lysosome
catalase                 catalase activity,        response to oxidative    peroxisome, plasma
                         antioxidant activity      stress                   membrane
toll-interacting protein protein binding, signal   inflammatory response    cytoplasm
                         transducer activity
beta-glucan              bacterial binding         regulation of innate     extracellular region
recognition protein                                immune response
manganese superoxide MnSD activity,                response to oxidative    mitochondrial matrix
dismutase                antioxidant activity      stress
plancitoxin              deoxyribonuclease II      DNA catabolic process,   lysosome
                         activity                  DNA binding




                                                                             Lindsay Braun and Cullen Taplin
TOLLIP   TOLLIP
Clams




        Roxanna Smolowitz
Differential Display
Serine Protease Inhibitor

• Critical factor in disease pathogenesis

• Serine proteases produced by pathogen
  •   Facilitate penetration

  •   Stimulate degradation of host proteins involved
      in immune defense
Expression
0.009
0.008
0.007
0.006
0.005
0.004
0.003
0.002
0.001
   0
        MA +   MA -    FL +            FL -   NJ +   NJ -
                              Strain
 Clams from different location of origin (MA, FL, NJ) all
 grown in common field sites in MA. Plus (+) and minus
 (-) symbols indicate two locations within a harbor.
photo: Maille Lyons
Field Trials
                   100
                                     June
                                     August
Percent Infected



                    75



                    50



                    25



                     0
                         MA    FL    NJ
QPX gene expression
QPX Serine Protease
Top Blastx Hit                                    Species associated with Blast Hit
                                                                            e-value %   similar
Glutathione-dependent formaldehyde-activating, GFARalstonia metallidurans    1.00E-04    45
Subtilisin-like serine protease pepC precursor    Aspergillus niger          2.00E-16    63
Permeases of the major facilitator superfamily    Yersinia frederiksenii         0.66    47
40s ribosomal protein s23                         Theileria annulata         9.00E-38    96
ATP-binding cassette / transporter, sub-family G, Toxoplasma gondii
                                                  member 4                   4.00E-25    60
mitogen-activated protein kinase                  Pan troglodytes           3.30E+00     46
Multidrug Resistance Protein family member (mrp-1)Caenorhabditis elegans     3.00E-14    66
mitogen-activated protein kinase 2                Toxoplasma gondii          1.00E-12    84
haemagluttinin repeat protein                     Pseudomonas fluoresens    2.70E+00     52
polysaccharide biosythesis protein                Methylococcus capsulatus  4.30E+00     47
Potassium inwardly-rectifying channel             Homo sapiens               1.00E-07    55
Relative Gene Expression
Relative Gene Expression




                                                                                             Optimal      Clam Tissue      Clam Tissue
                                                                                              Media       and Water         and Media
                                                                                                   Culture Conditions (ATTC strain)




                           ATTC     ATTC       Env_66                               Env_66
                           21!      10!         21!                                  10!
                                  Strain / Temperature
thanks




         Smolowitz

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Shellfish Disease

  • 1. Immune response and disease tolerance in shellfish Steven Roberts University of Washington School of Aquatic and Fishery Sciences
  • 3. Stress • Harmful anthropogenic compounds • Temperature • Hypoxia • Harmful organisms Bacteria,Viruses, Parasites
  • 5. Innate Immune System • Present from birth • “Non-specific” - acts on many pathogens and does not show specificity within a pathogen type • Does not become more efficient on subsequent exposure to same pathogen
  • 6. 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
  • 8. Outline • Genes expressed in oyster hemocytes • Dermo disease and mechanical stress • Disease resistance in oysters (and abalone) • Clams and QPX
  • 9. cDNA Library Rick Goetz
  • 10.
  • 12.
  • 13.
  • 14.
  • 15. gene expression level (fold increase over minimum)
  • 16. Dermo disease and mechanical stress Maps: Google - Oyster photos: G. Burreson
  • 17. Dermo disease and mechanical stress Gary Wikfors and Inke Sunila
  • 18.
  • 20. Resistance ? MVineyard flickr | cpurrin1 Roxannna Smolowitz Rick Karney
  • 21.
  • 22.
  • 23.
  • 24.
  • 25.
  • 26. Another example Dale Leavitt, Marta Gomez-Chiarri
  • 32. Mining ESTs from similar species Gene Molecular Function Biological Process Cellular Component rab1 GTPase activity vesicle-mediated golgi apparatus transport rab7 (WSSV receptor) GTPase activity, protein intracellular protein golgi apparatus, binding transport lysosome catalase catalase activity, response to oxidative peroxisome, plasma antioxidant activity stress membrane toll-interacting protein protein binding, signal inflammatory response cytoplasm transducer activity beta-glucan bacterial binding regulation of innate extracellular region recognition protein immune response manganese superoxide MnSD activity, response to oxidative mitochondrial matrix dismutase antioxidant activity stress plancitoxin deoxyribonuclease II DNA catabolic process, lysosome activity DNA binding Lindsay Braun and Cullen Taplin
  • 33. TOLLIP TOLLIP
  • 34. Clams Roxanna Smolowitz
  • 35.
  • 36.
  • 38. Serine Protease Inhibitor • Critical factor in disease pathogenesis • Serine proteases produced by pathogen • Facilitate penetration • Stimulate degradation of host proteins involved in immune defense
  • 39. Expression 0.009 0.008 0.007 0.006 0.005 0.004 0.003 0.002 0.001 0 MA + MA - FL + FL - NJ + NJ - Strain Clams from different location of origin (MA, FL, NJ) all grown in common field sites in MA. Plus (+) and minus (-) symbols indicate two locations within a harbor.
  • 40.
  • 42. Field Trials 100 June August Percent Infected 75 50 25 0 MA FL NJ
  • 43.
  • 45. QPX Serine Protease Top Blastx Hit Species associated with Blast Hit e-value % similar Glutathione-dependent formaldehyde-activating, GFARalstonia metallidurans 1.00E-04 45 Subtilisin-like serine protease pepC precursor Aspergillus niger 2.00E-16 63 Permeases of the major facilitator superfamily Yersinia frederiksenii 0.66 47 40s ribosomal protein s23 Theileria annulata 9.00E-38 96 ATP-binding cassette / transporter, sub-family G, Toxoplasma gondii member 4 4.00E-25 60 mitogen-activated protein kinase Pan troglodytes 3.30E+00 46 Multidrug Resistance Protein family member (mrp-1)Caenorhabditis elegans 3.00E-14 66 mitogen-activated protein kinase 2 Toxoplasma gondii 1.00E-12 84 haemagluttinin repeat protein Pseudomonas fluoresens 2.70E+00 52 polysaccharide biosythesis protein Methylococcus capsulatus 4.30E+00 47 Potassium inwardly-rectifying channel Homo sapiens 1.00E-07 55
  • 46. Relative Gene Expression Relative Gene Expression Optimal Clam Tissue Clam Tissue Media and Water and Media Culture Conditions (ATTC strain) ATTC ATTC Env_66 Env_66 21! 10! 21! 10! Strain / Temperature
  • 47.
  • 48. thanks Smolowitz