Mackenzie Gavery, Sam White, Steven RobertsUniversity of Washington, School of Aquatic and Fishery Sciences

Prostaglandins in the Pacific oyster:Investigations into form and function

Isolation and Characterization of Molecules

Prostaglandins are a group of lipid compounds that function as powerful biological signaling molecules. Prostaglandins are derived from arachidonic acid via a series of enzymatic reactions. Although prostaglandin pathways have been well characterized in vertebrates, information on the functional role in bivalves is lacking. The objectives of this study were to:

1) characterize the molecules involved in prostaglandin activity and

2) identify functional roles of prostaglandins in Pacific oysters.

Project Summary

Partial sequence obtained for Pacific oyster from EST(GenBank#: EW777722)

Previous research has suggested the presence of prostaglandins in bivalves. ELISA techniques were used to identify prostaglandin in Pacific oyster hemolymph.

Prostaglandin E2Full length sequence isolated.AA: 588 MW: 67.65 kDa

Cyclooxygenase (COX)

PGE2 Receptor

PGH2

Arachidonic Acid

Prostaglandin E2 (PGE2)

Plasma Membrane

Prostaglandin E2

Receptor

Phospholipase A

Cyclooxygenase

PGE Synthase

COOH

OH

O

COOHOO

COOH

Conclusions

Vertebrate COX-1

Vertebrate COX-2 COX A

COX B

COX

15s-COX

A full length cyclooxygenase sequence was obtained using Rapid Amplification of cDNA Ends based PCR. The deduced amino acid sequence is most similar to cyclooxygenase in soft coral. While two forms of cyclooxygenase have been identified in coral and vertebrates, only one was isolated from the oyster. Two partial sequences were identified in databases from other molluscs. RT-PCR analysis indicates cyclooxygenase is expressed primarily in gill and mantle.

To evaluate the role of prostaglandins in Pacific oyster immune response, Vibrio tubiashii challenges were performed in vivo and in vitro. RT-qPCR was used to evaluate gene expression of the PGE2 receptor in hemocytes. Circulating PGE2 levels were measured via ELISA. PGE2 receptor expression was upregulated after exposure to V. tubiashii indicating that prostaglandins are involved in the immune response of Pacific oysters.

Immune Function: Vibrio tubiashii Challenges

PGE2 Receptor PGE2 in Hemolymph

• Isolation of full-length cyclooxygenase sequence in Pacific oyster; the first reported in any bivalve species.

• Partial PGE2 receptor sequence identified, regulated upon pathogen exposure.

• Prostaglandin E2 detected and levels indicate role in immune system signaling.

• Future work includes developing these molecules as biomarkers for ecosystem health.

RT-PCR analysis indicates expression of the PGE2 receptor is highest in gill and mantle tissues. The partial sequence (EW777722) is most similar to the EP4 subtype in Danio rerio (36.2% similarity) and Homo sapiens (41.6% similarity). The EP4 subtype is involved withcAMP signaling.

C. gigasD. rerioH. sapiens

C. gigasD. rerioH. sapiens

C. gigasD. rerioH. sapiens

C. gigasD. rerioH. sapiens