Grantee Research Project Results
1999 Progress Report: Rapid Assessment of Coral Stress Using Gene Expression
EPA Grant Number: R827105Title: Rapid Assessment of Coral Stress Using Gene Expression
Investigators: Snell, Terry W.
Institution: Georgia Institute of Technology
EPA Project Officer: Hahn, Intaek
Project Period: October 1, 1998 through October 1, 2001
Project Period Covered by this Report: October 1, 1998 through October 1, 1999
Project Amount: $299,273
RFA: Exploratory Research - Environmental Biology (1998) RFA Text | Recipients Lists
Research Category: Biology/Life Sciences , Human Health , Aquatic Ecosystems
Objective:
There is widespread agreement that the health of coral reef systems worldwide are in a state of decline. The exact causes of this apparent decline is not known due to the variety of symptoms, including coral diseases, reduced coral growth, bleaching in corals and other photo synthetic mixotrophs, and impaired coral recruitment. Nutrients and toxins from point and nonpoint sources, and sediments from coastal watersheds are suspected to be the primary anthropogenic agents of reef health decline.At present, coral reef assessments are generally limited to evaluations of community structure and dynamics. These assessments are limited in that they cannot provide cause and effect information regarding community conditions, and by the time quantifiable community level changes occur, the damage to the reef has been done. Analytical techniques are needed that: (1) provide early warning of impending impacts to coral health; (2) are relatively fast (short duration), low cost, and that do not require a high level of technical sophistication; (3) quantify sub-lethal responses to pollutants and other environmental stressors; and (4) relate directly to coral biology and physiological responses. Our main objective in this work is to develop methods for rapid toxicity assessment using gene expression in stony corals.
Progress Summary:
Collection of Corals in the Florida Keys. We visited the Keys during April 1999, and exposed Acropora cervicornis to mercury, cyanide, the organophosphate mosquitocide Naled (Dibrom), naphthalene, and temperature stress. The mRNA from these samples was extracted, cleaned, and prepared for differential display PCR. Differentially expressed genes in response to these stressors were identified using differential display PCR and probes were prepared. The crossreactivity of the new probes with mRNA from Acopora exposed to other toxicants was tested and their specificty characterized. There are probes for six stressors, and these will provide the tools for investigating stressor-induced changes in gene expression in field populations.We visited the Keys for a second time during August 1999, and collected four additional species: Porites divaricata, Agaricia agaracities, Sidasteria radians, and Montasrea faveolata. These corals were exposed to mercury, dibrom, and napthalene. The mRNA from these samples was extracted and purified. This RNA from over 300 samples now is ready for hybridization with our Acropora probes that we have developed for six toxicants: permethrin, copper, dibrom, mercury, naphthlene, and cyanide. The crossreactivity of these probes developed for Acropora cervicornis with mRNA from other coral species will provide insight into how broadly applicable our probes are likely to be.
Differential Display of Toxicant-Induced Gene Expression in Acorpora Corals. We have developed techniques to extract and purify mRNA from the coral Acropora cervicornis, obtaining 1?2 mg in excellent condition for differential display PCR. These mRNAs have been reverse transcribed and used in differential display PCR with the commercially available kit RNAmap? from GenHunter Corp. Using several primer sets, we have identified 25 genes whose expression was increased after permethrin (insecticide) exposure. Similar PCR has been performed for copper-exposed A. cervicornis. We have identified 21 genes whose expression is upregulated upon copper exposure. From these, we have developed three probes for permethrin exposure based on genes that are upregulated. Likewise, we have developed three probes for copper exposure, two for upregulated and one for downregulated genes. The response of these genes to permethrin or copper exposure was quantified in replicate dot blots.
We have explored the crossreactivity of the probes, testing permethrin probes on copper-exposed corals, and vice versa. The purpose of this experiment was to determine the toxicant specificity of the probes. The probes also have been tested on another coral species, Tubastrea sp., a cup coral that is azooxanthallic (lacks algae symbionts). The binding of these probes to Tubastrea suggests that we have isolated coral genes, rather than genes of the dinoflagellate zooxanthallic symbiont Symbiodinium. We repeated this experiment using DNA extracted from Symbiodinium to confirm that the cDNA probes we have do not bind to algal genes, but are in fact coral in origin. Dot blots with these results are currently being analyzed. We visited the Keys for a second time during the last week of March. We performed another experiment in which we exposed A. cervicornis to mercury, the organophosphate mosquitocide Naled, naphthalene, and temperature stress. These samples have had mRNA extracted, cleaned, and prepared for differential display PCR. Differentially-expressed genes in response to these stressors will be identified and probes prepared. The crossreactivity of the new probes will be tested and their specificty characterized. When completed, there will be probes for six stressors, and these will provide the tools for investigating stressor induced changes in gene expression in field populations.
Collection and Extraction of Genomic DNA From Acropora Zooxanthellae. The probes also have been tested on DNA extracted from the zooxanthellae Symbiodinium to confirm that the cDNA probes do not bind to algal genes, but are in fact coral in origin. Dot blots have revealed that all but one of the genes detected by the probes are present in the coral. One is present only in the zooxanthellae, and the remaining are in the zooxanthellae as well as the coral. We will continue these experiments as new probes are developed to determine the origin of the genes we are sampling.
Future Activities:
In the following year, we expect to continue probe development until we have at least one reliable upregulated probe for each of the eight toxicants investigated. Upregulation will be quantified in slot blots using the DIG labeling system. Crossreactivities of each probe will be characterized to determine its specificity.Journal Articles on this Report : 1 Displayed | Download in RIS Format
Other project views: | All 5 publications | 1 publications in selected types | All 1 journal articles |
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Morgan MB, Vogelien DL, Snell TW. Assessing coral stress responses using molecular biomarkers of gene transcription. Environmental Toxicology and Chemistry 2001;20(3)537-543. |
R827105 (1999) R827105 (2000) |
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Supplemental Keywords:
water, corals, ecological effects, pesticides, metals, aquatic, coastal, ecology, genetics, marine biology, Florida Keys, southeast, biomarkers., RFA, Scientific Discipline, Geographic Area, Ecosystem Protection/Environmental Exposure & Risk, Aquatic Ecosystems & Estuarine Research, Ecosystem/Assessment/Indicators, Ecosystem Protection, Oceanography, Aquatic Ecosystem, Ecological Effects - Environmental Exposure & Risk, Environmental Microbiology, Southeast, Ecological Risk Assessment, ecological exposure, anthropogenic stress, anthropogenic stresses, coral reef ecosystem, monitoring, risk assessment, toxicity studies, adverse impacts, coral reefs, stressors, Florida Keys, genes, natural stressors, RNA, toxicity, aquatic ecosystems, coral reef communities, aquatic ecology, anthropogenic pollutant effectsRelevant Websites:
http://www.biology.gatech.edu/snell.htmlProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.