ECOTOXICOGENOMICS of DAPHNIA MAGNARathana Yim, Don Pham, Hun-je Jo, Leona Scanlan, Chris Vulpe

ABSTRACT

INTRODUCTION

SYSTEMS UNDER STUDY

RESEARCH and METHODS RESULTS

DISCUSSION

Parthogenic, therefore; Uniform GenomeShort lifespan and generation

Broods many clone offspringIdeal organism for studying toxicity

Freshwater zooplankton

Br

Br

Br

Br

Br

BrBr

Br

(Brominated Flame Retardant)

EMERGENT CONTAMINANT

EMERGENT CONTAMINANTS BIOACCUMULATES

PHARMACEUTICALS

Word press 2008Yosemite 2009

a. b. c. d. e.

Figure 1. (1a-1e) Necessary preparation for RNA extraction

PREPARATION FOR DAPHNID EXPOSURE

RSC publishing 2008RSC publishing 2008 L. Hernandez 2009

a. Gather Parthogenic Female D. magnab. U.S. EPA Standard for Culturing D. magnac. Acute Toxicity test for Lethal Concentration (LC 10, LC 50)

d. Toxicity Bioassay with Toxicant (Cadmium)e. 24 hours, 48 hours Exposure to Toxicant (Cadmium)

GENOMIC TECHNOLOGY

1 Week Old D. magna

Figure 2. (2a-2e) RNA extraction via Homogenization to Hybridization

2a. Exposed Daphnias (1 Week Old)2b. Homogenization of D. magna2c. Run Gel electrophoresis for mRNA bands

2d. Gel will give result of bands (mRNA)2e. Hybridization of RNA sample for Microarray

2a.

NIH Gov 2009

D. Pham 2009 Davidson 2009D. Pham 2009

2b. 2c. 2d. 2e.

Figure 4. Microarray analysis of DEG of D. magna, dots above the blue line represents control expressed genes, while the dots below represents the exposed expressed genes.

Figure 3. Microarray of DEG of D. magna exposed to Cadmium

In this figure, points above and below the normalization line (blue line) represent the under- and over- expression of the D. magna genes when exposed to Cd, respectively. The majority the dots that reside along the line show that those genes are being expressed in both the control and exposed group.

Transcripts of the exposed group were labeled with Cy5 dye (red) and transcripts of the control group were labeled with Cy3 dye (green). Red dots show over- expression of the gene and green dots show underexpression of a gene when exposed to cadmium.

ACKNOWLEDGEMENTS

LITERATURE REVIEWED

Analyzing differentially expressed genes (DEG) in aquatic organisms is a useful technique to finding biomarkers of exposure. It can provide a more comprehensive and diagnostic approach to monitor emergent contaminants in the environment. When the organisms are exposed to certain toxicants, their gene expression profile yields a specific set of differentially expressed genes for each compound. In this research, we exposed the water “flea” known as Daphnia magna to high priority pollutants and identified the DEG for each toxicant using micro array technology. Results show that the group of D. magna exposed to cadmium sulfate exhibits a unique set of DEGs. In the future, the DEG profile can be used to deduce cadmium’s toxic mode of action or for exposure assessment. Studying the DEG of D. magna can help us understand how these compounds affect aquatic organisms and ecosystems and may someday provide a detailed understanding of how these chemicals affect the biological pathway of organisms in constant exposure to emergent contaminants.

Control D. magna Exposed D.magna Cd

mRNA extracted from cell

Reverse Transcription fluorescently labeled with

Cy3 (Green) and Cy5 (Red)

Cy3 (Control Daphnid) Cy5 (Daphnid exposed to Cadmium)

Combine equal amount and hybridize onto micro

array

cDNA microarray

Scan

Exposed toxicant micro array for Differentially

Expressed Genes (DEG) in D. magna

2+Table. Microarray Legend

Both Control and Exposed Genes Expressed

Control Genes Expressed

Exposed Genes Expressed

HYBRIDIZATION to MICROARRAY Based on our research, we discovered that D.magna exposed to cadmium yielded a specific set of differentially expressed genes (DEGs). We can use the DEGs of the exposed D. magna to uncover novel biomarkers to detect the presence of pollutants in freshwater biomes of these organisms. In future studies, we will want to verify the specificity of DEGs to cadmium by testing our methods in different laboratory settings and also the aquatic environment. We also will want to determine the functions of the DEGs and deduce the toxic mode action of the chemical.

Control A Control B Control C Exp A Exp B Exp C

RNA samples show two bands (proteins that transcribe DNA to mRNA) which means the samples are good and ready for hybridization and microarray analysis.

Poynton, Helen; C. and Chris Vulpe. “Ecotoxicogenomics: Emerging Technologies for Emerging Contaminants.” Journal of the American Water Resources Association (AWRA). Vol 45. 1. 2009 February

Poynton, Helen; C. Wintz, Henri, and Chris Vulpe. “Progress in ecotoxicogenomics for environmental monitoring, mode of action, and toxicant identification.” Advances in Experimental Biology: Volume 2. 2008.

Benzie, John A. H; “The genus Daphnia including Daphniopsis, Anomopoda: Daphniidae.” Guides to the Identification of the Microinvertebrates of the Continental Waters of the World. Backhuys Publishing. Copyright 2005.

Dr. Chris Lever, Dr. Sang Lee, Lynn Huntsinger, Sally Fairfax, and Dr. Keith Gilless.

The Environmental Leadership Pathway program and the National Science Foundation for blessing me with this oppurtunity.

Dr. Chris Vulpe, Don Pham, Hun-je Jo, Leona Scanlan, Candace Clark, Daniel Nowinski, Ashneel Krishna, Luis Hernandez, Camice Revier, and Jose Albarracin.

Poynton, Loguinov, Alexandre; Varshavsky, Julia; Chan, Sarah; and Vulpe, Chris. “Gene Expression Profiling in Daphnia magna Part I: Concentration-Dependent Profiles Provide Support for the No Observed Transcriptional Effect Level.” Environmental Science and Technology.

From the acute 48-hr acute toxicity bioassay, we found the LC 50 used for moderately hard water. The LC 50 value correlates to that of 24 hr which is 180 ug/L. We found that at this LC 50, the D. magna exposed at this low level concentration yielded optimal lethality in determining gene expression.

Department of Nutritional Sciences and Toxicology; University of California, Berkeley

Emergent contaminants or unregulated chemicals are slowly leeching into the ecosystem through human excretion, disposition of pharmaceuticals, and pollution (Poynton 2009). Traces of chemicals left behind are accumulating within freshwater biomes, which in turns bio-accumulates within streams, rivers, and lakes. The study of pollutants and its effect to the environment has been considered one of the biggest concerns for environmentalists, scientists, and biologists; due to the fact that unforeseen effects of accumulation of these chemicals in the ecosystem may rise to toxic levels with detrimental effects on organisms on all trophic levels (Poynton 2008). The risk of these compounds to aquatic ecosystems has yet to be fully understood. New technology has not yet been developed to detect these chemicals; however, a more strategic approach of using genomics and DNA technology is paving the way for a more comprehensive diagnostic approach. The use of aquatic crustaceans, D. magna and its genome is ideal to understand how these compounds affect aquatic ecosystems.

Pharmacetix 2009