Grantee Research Project Results
2000 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, 1999 through October 1, 2000
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 photosynthetic mixotrophs, and impaired coral recruitment. Nutrients and toxics 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 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 August 2000, and exposed Acropora cervicornis to the organophosphate mosquitocide Naled (Dibrom) in bags in situ on Admiral Reef. The mRNA from these samples was extracted, cleaned, and prepared for slot blotting to detect upregulation of stress induced genes. This is the first time that we tried our probes on coral RNA samples collected in the field. Small coral fragments were placed in 5 L bags underwater on the reef. These bags were either controls receiving no further treatment, or they were injected with 0.5 mg/L Dibrom and the corals exposed for 4 hours. Coral fragments were removed and placed in Trizol for later extraction of RNA. These samples were compared to samples collected from June 1997, April 1999, and August 1999.Characterizing Probe Responsiveness. Our two dibrom probes are responsive to different stressors. The long probe (550 nucleotides) detected genes that were induced by 4 hour exposures to 0.5 and 5 µg/L dibrom in the laboratory (Figure 1). In addition, this probe detected a gene induced by exposure to 300 µg/L naphthalene. This gene is likely to be the same or very similar to the dibrom stress gene due to the specificity of complimentary nucleotide binding. In contrast, this long dibrom probe did not detect any newly synthesized mRNAs in response to lindane, permethrin, copper, or mercury exposures. These samples of A. cervicornis were collected in April 1999, near Tennessee Reef in the middle Florida Keys when ambient water temperature was 26?C. There is no expression of this dibrom induced stress gene in the field samples that served as controls (Figure 1).
Figure 1. Array using the long dibrom probe. One µg samples of RNA extracted from A. cervicornis exposed to toxicants and blotted on a nitrocellulose membrane. All toxicant exposures were for 4 hours at the concentrations indicated. BC ? boat control, RNA extracted immediately after collection without toxicant exposure; LC ? lab control, RNA extracted after returning to the lab (about 6 hours after collection); AC ? acetone control, RNA extracted in lab after 4 hours exposure to acetone (solvent control). The darkness of the slot blot is directly proportional to the amount of probe binding.
Besides the dibrom exposures in lab, we exposed A. cervicronis samples to temperatures of 25?, 30?, and 35?C. Direct transfer from ambient to 25?C or 30?C induced the expression of a temperature responsive gene (Figure 2). Transfer to 35?C caused immediate coral bleaching and reduced expression of the temperature responsive gene. This is expected because heat stress was so great at 35?C that coral cells were unable to carry on normal mRNA synthesis. Densitometry was performed on all the slot blots, providing quantitative measures of the amount of up-regulation in gene expression.
Figure 2. Temperature responsiveness of the coral gene detected by the long dibrom probe. Controls are samples exposed to ambient temperatures in the lab or on the collecting boat. Four hour lab exposed corals were transferred directly from 26?C ambient temperature in April 1999 to the three experimental temperatures. The field sample was collected in August 1999, when ambient water temperature was 31?C.
Comparison of field samples collected of A. cervicornis from the Florida Keys over 3 years indicates a temporal pattern of stress induced gene expression. The first collections were made in June 1997, when ambient water temperature was 29 C (Figure 3). No stress gene expression in this sample was detected by the long dibrom probe. Likewise, samples collected in April 1999, with an ambient temperature of 26?C showed no expression of this stress gene. In contrast, samples collected on this date and exposed to dibrom in the lab demonstrated stress gene induction. Field samples collected in August 1999 and 2000, at an ambient temperature of 31?C both exhibited induced gene expression. This suggested to us that these samples were experiencing stress in the field, but what kind of stress was it? Temperature stress was an obvious candidate because this probe is known to be responsive to temperature. The gene expression detected by this probe also is induced by dibrom and naphthalene exposure, so perhaps these populations of A. cervicornis were exposed to organophosphate pesticides or polycyclic aromatic hydrocarbons. Exposure to copper, mercury, and the organochlorine pesticides permethrin and lindane could not explain this gene expression pattern. An accurate diagnosis of the stressors required data from more probes.
Figure 3. Induction of the coral gene detected by the long dibrom probe in field samples. 6/97, 4/99, 8/99, and 8/00 refer to A. cervicornis samples collected in June 1997, April 1999, August 1999, and August 2000 at the ambient water temperatures indicated. Strong gene induction is visible only in the latter two samples. As reference, a sample induced by dibrom exposure in the lab is shown.
These data should provide scientific insight into the major stressors impacting corals in the Florida Keys National Marine Sanctuary. Using multiple probes and biweekly sampling, we should be able to detect the onset of stress in corals and implicate the major stressors.
Future Activities:
In the next year, we expect to continue to characterize probe responsiveness to all of the eight toxicants investigated. We expect to develop four new probes induced by UV, salinity, shading, and sediments using subtractive hybridization. Upregulation will be quantified in slot blots using the DIG labeling system. Cross-reactivities 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, UV, sediments, salinity, aquatic, coastal, ecology, genetics, marine biology, Florida, FL, 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.