Analysis of Genes Isolated from Plated Hemocytes

of the Pacific Oyster, Crassostrea gigas

Steven RobertsSchool of Aquatic and Fishery Sciences

University of Washington

Rick Goetz Great Lakes WATER Institute

University of Wisconsin

Goal

Better understand stress response

Laura Canesi, Gabriella Gallo, Miriam Gavioli, and Carla PruzzoBacteria–hemocyte Interactions and Phagocytosis in MarineBivalves. 2002. Microscopy Research And Technique 57:469–476

Immune System

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

Pacific OysterC. gigas

Incubate 24 hours Wash in sterile seawaterExtract with Tri-Reagent

+ sterilesaltwater

Plated hemocyte cDNA library

Plated hemocyte cDNA library

Prior to washing After washing

Gene - DNA

Transcript - mRNA

Extract from tissue

Make cDNA

Clone into a “Vector”

Randomly sequence clones = “Expressed Sequence Tags”

(ESTs)

ESTs

(Complementary DNA = Copy)

= cDNA Library

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 AntiproteasesGene Function

Proteases and AntiproteasesGene Function

Proteases and AntiproteasesGene Function

Inhibitors

Proteases and AntiproteasesGene Function

Cathepsins

Proteases and AntiproteasesGene Function

Cathepsins

Proteases and AntiproteasesGene Function

C. virginica Homologs

Proteases and AntiproteasesGene

C. virginica Homologs

Receptors and other proteinsGene Function

Prostaglandin E2EP4 subtype

Prostaglandin

15-hydroxyprostaglandin dehydrogenase

PGE2 breakdown

lipoxygenases production of leukotrienes,

lipoxins,hydroperoxy fatty acids

other ESTs present

Prostaglandin

Mackenzie Gavery - Poster

Receptors and other proteinsGene Function

Ectodysplasin A2Receptor

Transcription / Translation / Cell CycleGene Function

Erythroid differentiation-related factor

Transcription / Translation / Cell CycleGene Function High-mobility

group proteins

Transcription / Translation / Cell CycleHigh-mobilitygroup proteins

Transcription / Translation / Cell CycleHigh-mobilitygroup 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