Grantee Research Project Results
Final Report: Sediment Contaminant Effects on Genetic Diversity: New Approach using DNA Analyses of Meiobenthos
EPA Grant Number: R825439Title: Sediment Contaminant Effects on Genetic Diversity: New Approach using DNA Analyses of Meiobenthos
Investigators: Coull, Bruce C. , Quattro, Joseph M. , Chandler, G. Thomas
Institution: University of South Carolina at Columbia
EPA Project Officer: Aja, Hayley
Project Period: February 24, 1997 through February 23, 2000 (Extended to February 23, 2001)
Project Amount: $372,642
RFA: Exploratory Research - Environmental Biology (1996) RFA Text | Recipients Lists
Research Category: Aquatic Ecosystems , Biology/Life Sciences
Objective:
The objective of the research project is to develop a population-genetics level bioassay that is more sensitive and more relevant to long-term benthic community protection than are current U.S. Environmental Protection Agency (EPA) acute, chronic, and sub-chronic toxicity test standards. Chronic toxicant loads in sediments may drive populations toward a limited set of tolerant genotypes over time. Such reductions in genetic diversity reduce a population's plasticity to adapt to the new stresses that can arise as anthropogenic stress on aquatic ecosystems increase.Research was structured to analyze the genetic variation in Microarthridion littorale across its southeastern range. M. littorale (Poppe, 1881), as currently described, is the most abundant and cosmopolitan of harpacticoid copepod species, worldwide. After this assessment of its genetic history, or phylogeography, the project focused on two different aspects of the genetic diversity in the copepod populations: (1) variation in mitochondrial DNA haplotypes and its correlation to environmental pollution history, and (2) response of a mixed genetic stock to laboratory exposure to pesticides.
Summary/Accomplishments (Outputs/Outcomes):
One of the first obstacles to genetic research involves extracting high quality DNA from specimens, a problem compounded when the organism of interest is small. A method was developed in this grant that allows for good template DNA for PCR and repeated amplifications from a single individual (< 0.5 mm in length). It is not automated, but allows for application to population-level genetic investigations and was used extensively in the course of genetic investigation for this grant.Part of the preliminary work for this research was to understand the geographic population genetic history of the harpacticoid copepod Microarthridion littorale (Poppe, 1881). Genetic relationships among populations (southeast Atlantic and northern Gulf coast, USA) were estimated using sequence data from two loci: one mitochondrial [cytochrome b (Cyt b)] and one nuclear [first internal transcribed spacer of ribosomal DNA (ITS-1)]. Copepods were collected from seven estuaries in 1997/1998. Allelic phylogenies constructed from both genes were generally in agreement (or concordant) and suggested that M. littorale populations are genetically structured over large geographic scales (hundreds of kilometers). Three well-supported groups were found in both gene trees comprising phylogenetic groups (or clades) of alleles sampled from South Carolina, Florida, and Louisiana. Alleles from the Savannah, Georgia, sample formed a single group using the Cyt b data, but this cluster was not distinguishable with comparable ITS-1 data. A single specimen from Louisiana was classified in different clades depending on the locus assayed.
The focus then was to characterize the population genetic structure of Microarthridion littorale from parts of North Carolina, South Carolina, and Georgia, USA. Populations were investigated by a geographic survey of a 348-base pair fragment of the mitochondrial cytochrome b gene. DNA sequence divergence among 198 individuals was less than 4.3 percent, but three divergent mitochondrial lineages were found that differed by six to nine unique nucleotide changes. A rapid assay was developed to distinguish among mitochondrial lineages for which an additional 333 specimens were surveyed. The three lineages co-occurred in seven out of ten sampling sites. Data analyses were carried out separately for individuals assayed by DNA sequencing as well as a combined data set that included individuals typed by restriction endonuclease digestion. An analysis of molecular variance (AMOVA) indicated that a significant proportion of the total genetic variance could be partitioned among populations, although no significant correlation between geographical and genetic distance was uncovered. Distribution of these haplotypes appeared to be more related to the pollution history of a site, rather than its geographical proximity to other sites. Specifically, the frequencies of two haplotype groups are greatly diminished and sometimes absent in contaminated South Carolina tidal creeks, while the third is omnipresent.
In a detailed laboratory study of these haplotypes, representatives of M. littorale with the three haplotype groups were collected from sediments of an estuarine creek containing low to undetectable levels of toxicants, and then exposed to a toxic (LC90) aqueous mixture containing an organophosphate (chlorpyrifos) and organochlorine pesticide (DDT, mixed isomers). Haplotype frequencies among the survivors of the exposed animals were compared to those among the survivors of the control group. The haplotype group that was most frequent in populations from contaminated South Carolina estuaries survived exposure at a statistically higher frequency than did individuals in the control group. The frequencies of the three groups were not significantly affected by control conditions (i.e., group frequencies of randomized field-collected individuals equaled those surviving the control conditions). The differential survival of the haplotype groups in the laboratory pesticide mixture supports the observed field pattern that haplotype distribution is a result of contaminant history of the sites.
However, all contaminants do not affect copepod populations in the same way. In a separate test, Microarthridion littorale were collected from three South Carolina, USA, estuaries having different pollution stress histories (i.e., pristine sediments, high polycyclic aromatic hydrocarbon [PAH] sediments, high metals/moderate PAH sediments), and then assayed for survival and reproductive output in 14-day exposures to pristine and heavily PAH/metals-contaminated sediments. Whole-sediment reproduction bioassays were used to determine whether copepods exposed to a highly contaminated sediment mixture exhibited differential survival and reproductive outputs as a function of previous environmental histories, and whether genetic relatedness among populations measured as DNA sequences of the Cyt b gene were linked to copepod contaminant tolerance. Overall, adult survival and reproductive success in contaminated sediments were significantly reduced relative to controls for all three populations irrespective of environmental histories. Significant genetic differentiation, however, was found between copepod populations from the control and the two contaminated sites. Copepods present were coping with pollution specific only to their own habitat, and changes in this threat were as devastating as the original contaminant load to unexposed individuals. Cyt b appears to be serving as a marker that is not tightly linked to nuclear genes, which are probably under selection.
Conclusions:
Microarthridion littorale provides a good system for future exploration of genetic variation under the stress of pollution. The species within South Carolina is a separate entity from other genetic lineages described above, so it is unclear whether others will show similar or different responses to contaminants. The absolute mechanisms of contaminant resistance are not known, but we are expanding this work by isolating nuclear genes that may be under direct selection by pesticides or PAHs.Journal Articles on this Report : 5 Displayed | Download in RIS Format
Other project views: | All 11 publications | 6 publications in selected types | All 5 journal articles |
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Kovatch CE, Schizas NV, Chandler GT, Coull BC, Quattro JM. Tolerance and genetic relatedness of three meiobenthic copepod populations exposed to sediment-associated contaminant mixtures: role of environmental history. Environmental Toxicology and Chemistry 2000;19(4):912-919. |
R825439 (1999) R825439 (Final) R826399E02 (Final) |
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Schizas NV, Street GT, Coull BC, Chandler GT, Quattro JM. An efficient DNA extraction method for small metazoans. Molecular Marine Biology and Biotechnology 1997;6(4):381-383. |
R825439 (1999) R825439 (Final) |
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Schizas NV, Street GT, Coull BC, Chandler GT, Quattro JM. Molecular population structure of the marine benthic copepod Microarthridion littorale along the southeastern and Gulf coasts of the USA. Marine Biology 1999;135(3):399-405. |
R825439 (1999) R825439 (Final) R826399E02 (Final) |
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Schizas NV, Chandler GT, Coull BC, Klosterhaus SL, Quattro JM. Differential survival of three mitochondrial lineages of a marine benthic copepod exposed to a pesticide mixture. Environmental Science & Technology 2001;35(3):535-538. |
R825439 (1999) R825439 (Final) R825279 (Final) R826399E02 (Final) R827397 (2002) R827397 (Final) |
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Schizas N, Coull B, Chandler G, Quattro J. Sympatry of distinct mitochondrial DNA lineages in a copepod inhabiting estuarine creeks in the southeastern USA. Marine Biology 2002;140(3):585-594. |
R825439 (1999) R825439 (Final) R826399E02 (Final) R827397 (2002) R827397 (Final) |
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Supplemental Keywords:
copepod, estuary, water, exposure, bioavailability, vulnerability, animal, aquatic, habitat, conservation, zoology, South Carolina, Georgia, Florida, Louisiana, SC, GA, FL, LA., RFA, Scientific Discipline, Toxics, Water, Waste, Geographic Area, Ecosystem Protection/Environmental Exposure & Risk, Water & Watershed, Bioavailability, Ecology, Ecosystem/Assessment/Indicators, Ecosystem Protection, exploratory research environmental biology, Chemical Mixtures - Environmental Exposure & Risk, Contaminated Sediments, Environmental Chemistry, Genetics, pesticides, State, Chemistry, Ecological Effects - Environmental Exposure & Risk, Zoology, chemical mixtures, Ecological Effects - Human Health, Watersheds, Ecological Indicators, ecological exposure, habitat, sediment bioassay, aquatic ecosystem, genetic sequencing, contaminant transport, sediment contaminant effects, vulnerability, contaminated sediment, DNA analyses of meiobenthos, Georgia (GA), chemical contaminants, polymerase chain reaction, Louisiana (LA), ecological impacts, superfund site, aquatic ecosystems, bioassay, DNA, South Carolina (SC), pesticide runoff, Florida, FL, meiobenthos, exposure assessment, genetic diversity, DNA analysisRelevant Websites:
http://www.biol.sc.edu/~coull_lab/
http://enhs.sph.sc.edu/faculty/Chandler/chandler.html
http://www.mtsu.edu/meio/
Progress 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.