safs gss nov 2008: characterization of prostaglandins in the pacific oyster crassostrea gigas:...

Characterization of prostaglandins in the Pacific oyster Crassostrea gigas: evidence for a role in immune response

Mackenzie Gavery, M.S. StudentRoberts Lab

Molecules with Diverse Functions•Vertebrates

• Mediate swelling and inflammation

• (COX inhibitors)

• Inducing fever

• Maintaining pregnancy/reproductive timing

• Regulating ion transport

Molecules with Diverse Functions

• Invertebrates

• Ion transport

• Reproduction

• Immune Response

•recently discovered a gene encoding prostaglandin E2 receptor in Pacific oysters

• significantly upregulated after exposure to Vibrio tubiashii

Roberts et al

What are prostaglandins?•Eicosanoids: a family of biologically active

metabolites of C20 polyunsaturated fatty acids.

•Prostaglandins biosynthesis begins with the from arachidonic acid, an omega-6 fatty acid.

prostaglandin E2:

Arachidonic Acid Cascade

PGE receptor

Outline•Characterize molecules in prostaglandin

pathway

▫ID gene fragments using ESTs

▫Tissue distribution of oyster

▫Isolate complete ORF of oyster COX gene

•Challenge Experiments

•Applied research

Arachidonic Acid Cascade

PGE receptor

Sequences

PGE receptor

ESTs: snail, mussel

ESTs: oyster!,mussel•AM856036

ESTs: ongoing

ESTs: •EW777722

Data

PGE receptor

Full-length Sequence

PGE receptor

STATS:

•Size: 1700 bp

•pred. # introns: 10

•Amino acids: 587

COX Gene Similarities

Outline•Characterize molecules in prostaglandin

pathway

▫ID gene fragments from genomics database

▫Tissue distribution of oyster

▫Isolate complete ORF or oyster COX gene

•Challenge Experiments

•Applied research

ExperimentsVibrio tubiashii exposures

whole organism plated

hemocytes

Analytical Results

PGE receptor

• PGE (ELISA)

• Receptor (PCR)

Vibrio exposure-ELISA results

PGE conjugate

PGE sample

100% 0%

Gill Tissue/Vibrio– qPCR PGE receptor

Plated hemocytes/Vibrio - qPCR

Outline

•What are prostaglandins?

•Characterization of genes involved in biosynthesis of prostaglandins

•Experiments and Results

•Applied research

Applied Research

Physiological Processes:•Immune Response•Ion transport•Reproduction•Homeostasis

temperature

pathogen exposureacidification

pesticides

Applied Research

•Prostaglandins appear to play a role in the immune response of oysters.

•In vitro hemocyte cultures may be a good experimental model to study physiological responses to disruptions of this pathway

▫how would changes in environmental conditions (pollutants, temperature, pH) affect the regulation of arachidonic acid metabolism?

▫What physiological effects might this have?

Acknowledgements

•Advisor, Steven Roberts•Research scientist, Sam

White•Friedman and Seeb Lab