Development of genetic markers to assess disease resistance in the Eastern Oyster, Crassostrea virginica Analysis of differentially expressed transcripts Elie Diner - University of Rochester, New York Christina Romano and Steven Roberts - Marine Biological Laboratory

Introduction C. virginica is a marine bivalve of the phylum Mollusca with significant economic and environmental importance. Populations have realized significant declines due to a variety of factors including parasites Perkinsus marinus (Dermo) Artificially selected cultures and wild population subjected to heavy disease pressure demonstrate disease resistance.

C. virginica is a marine bivalve of the phylum Mollusca with significant economic and environmental importance.

Populations have realized significant declines due to a variety of factors including parasites Perkinsus marinus (Dermo)

Artificially selected cultures and wild population subjected to heavy disease pressure demonstrate disease resistance.

Purpose of Current Study Determine differences in gene expression patterns in Dermo resistant oysters What genes contribute to resistance?

Determine differences in gene expression patterns in Dermo resistant oysters

Experimental Design Characterize expression level of selected genes from 3 groups of oysters: Artificially selected line, proven Dermo resistant (Rutgers) Wild population - heavy disease pressure (Green Hill) Wild population – little disease pressure (Blue Bill) Determine level of Perkinsus infection and correlate with gene expression data

Characterize expression level of selected genes from 3 groups of oysters:

Artificially selected line, proven Dermo resistant (Rutgers)

Wild population - heavy disease pressure (Green Hill)

Wild population – little disease pressure (Blue Bill)

Determine level of Perkinsus infection and correlate with gene expression data

Purpose of Current Study Determine differences in gene expression patterns in disease resistant oysters Examine host-parasite interactions, focusing on differential gene expression between oyster groups.

Determine differences in gene expression patterns in disease resistant oysters

Examine host-parasite interactions, focusing on differential gene expression between oyster groups.

Gene Expression RNA was extracted from gill tissue (contains hemocytes), diluted to equal concentration and used in SYBR Green one step RT-PCR assays Genes quantified were selected based on function in other organisms and relation to immune response.

RNA was extracted from gill tissue (contains hemocytes), diluted to equal concentration and used in SYBR Green one step RT-PCR assays

Genes quantified were selected based on function in other organisms and relation to immune response.

Real-time RT-PCR CD646593 IK cytokine CD649275 IAP 4 CD649038 Bcl-X CD649164 Apoptosis Inh. Facto Accession # CD526707 MAPK8 int. prot. CD648711 Hsp 70 X75894 Actin

MAP8K Interacting Protein The MAP Kinase family is one of many groups of protein cascades responsible for regulating growth and cell cycle of an individual cell

Hemocyte population and Perkinsus Cell cycle regulation Hemocyte growth and proliferation could be modulated in response to Perkinsus Could indicate mechanism by which Perkinsus infects

Cell cycle regulation

Hemocyte growth and proliferation could be modulated in response to Perkinsus

Could indicate mechanism by which Perkinsus infects

BCL-X A protein that acts to suppress the apoptosis/programmed cell death pathway.

Apoptosis – BCL-X Resistant oyster strains could downregulate apoptosis suppression Allowing for increased apoptosis Decreasing number of hemocytes available for Perkinsus proliferation

Resistant oyster strains could downregulate apoptosis suppression

Allowing for increased apoptosis

Decreasing number of hemocytes available for Perkinsus proliferation

Wild Type Dermo Resistant Proposed Mechanism Under healthy conditions… Natural Hemocyte Renewal Programmed Cell Death

Wild Type Dermo Resistant Proposed Mechanism Dermo Infection Programmed Cell Death Natural Hemocyte Renewal BCL-X BCL-X

Hemocyte population and Perkinsus Cell cycle regulation Hemocyte growth and proliferation could be modulated in response to Perkinsus Could indicate mechanism by which Perkinsus infects Apoptosis Dermo resistant oysters have the ability to increase apoptosis, decreasing hemocytes available for Perkinsus growth and proliferation

Cell cycle regulation

Hemocyte growth and proliferation could be modulated in response to Perkinsus

Could indicate mechanism by which Perkinsus infects

Apoptosis

Dermo resistant oysters have the ability to increase apoptosis, decreasing hemocytes available for Perkinsus growth and proliferation

Perkinsus Quantitative RT-PCR Using genomic DNA extracted from mantle and a florescent probe for Dermo.

Summary Oyster strains appear to demonstrate different gene expression patterns that could explain ability to survive parasite infection Need to look closer at correlation of Dermo presence and gene expression in individual oyster In future, gene expression could be used for marker assisted selection of local broodstock that are Dermo resistant

Oyster strains appear to demonstrate different gene expression patterns that could explain ability to survive parasite infection

Need to look closer at correlation of Dermo presence and gene expression in individual oyster

In future, gene expression could be used for marker assisted selection of local broodstock that are Dermo resistant

Acknowledgements Steven Roberts Christina Romano Marta Gomez-Chiarri and Dale Leavitt for Oysters Grant funding from –NRAC USDA

Steven Roberts

Christina Romano

Marta Gomez-Chiarri and Dale Leavitt for Oysters

Grant funding from –NRAC USDA