2001 Progress Report: Genetic Diversity in California Native Fish Exposed to Pesticides

EPA Grant Number: R826603
Title: Genetic Diversity in California Native Fish Exposed to Pesticides
Investigators: Anderson, Susan L. , Kuivila, Katherine , May, Bernard , Wilson, Barry W.
Institution: University of California - Davis
EPA Project Officer: Packard, Benjamin H
Project Period: August 1, 1998 through January 31, 2003
Project Period Covered by this Report: August 1, 2000 through January 31, 2001
Project Amount: $649,003
RFA: Ecological Indicators (1998) RFA Text |  Recipients Lists
Research Category: Ecological Indicators/Assessment/Restoration , Ecosystems

Objective:

The overarching goal of this project is to determine the effect that pesticide exposure may have on genetic variation in a California native fish. The first objective is to examine populations of the Sacramento sucker (Catostomus occidentalis) exposed to landscape-scale pesticide releases to determine whether changes in genetic diversity are associated with indicators of pesticide exposure or natural genetic variation. Molecular techniques to be utilized include amplified fragment length polymorphisms (AFLP) and variation in microsatellite loci. The design of this study also includes a multi-level exposure assessment and the use of short-term biomarkers to elucidate pesticide exposure and potential genotoxic responses. The second objective is to compare the AFLP technique with the randomly amplified polymorphic DNA (RAPD) technique to determine which produces the most informative and reproducible DNA fingerprints. The RAPD technique is significant because it is used in ongoing EPA Environmental Monitoring and Assessment Program (EMAP) studies; yet, the newly available AFLP technique permits examination of more of the genome per unit effort and is reputed to be more reproducible. The third objective is to evaluate potential linkages between any observed changes in genetic variation and fitness parameters in individuals and populations. This study is unique because genetic variation associated with contaminant exposure has not been evaluated in fish populations on a large geographic scale.

Progress Summary:

Laboratory research for Objective 2 was completed 2 years ago, and a manuscript on the work was published in the journal Ecotoxicology. In this report, a revision of the project goals is being proposed: the level of effort under Objective 2 would be expanded and Objective 3 would be eliminated. This will allow for theimplications of the project findings to be addressed more directly to environmental managers. This year's progress relates mainly to Objective 1.

Objective 1—Population Genetics

In the past year, significant progress was made in microsatellite and AFLP screening of populations. The task of analyzing and interpreting marker data from approximately 1,000 fin clips collected at eight reference and four impacted stations in six watersheds is nearing completion. One strength of this program (described in detail last year) is the extensive sampling design that allows for a high level of field replication and a sophisticated exposure analysis.

Microsatellite loci are being used to assess the genetic structure and to detect new mutations in populations of the Sacramento sucker. Microsatellites are tandem repeats of short nucleotide motifs, and their alleles differ in the number of these repeat units. Microsatellites are considered one of the most powerful genetic markers because of their high variability and relative ease of scoring—alleles can be scored consistently and unambiguously across gels. Microsatellites are appropriate for the detection of new mutations because, as a result of their repeat motifs, they have a different form of mutation than point mutations found in other areas of a genome. It is hypothesized that this type of mutation is more affected by extrinsic mutagens than are point mutations. Extrinsic mutagens may change the characteristic way in which repeat units are generated—leading to alleles of much smaller or much larger size—and/or increase the number of new alleles in the population.

During the past year, protocols have been developed and optimized for collecting genotypes on the MJ-GeneSys BaseStation; 296 individual genotypes per gel can be run, and the process of data analysis for each gel has been streamlined. The best procedure for normalizing the size calling across gels is being determined, and single base pair differences are being resolved. One method that may help to normalize the size calling across gels is to run not only internal size standards, but also a same known individual on every gel.

DNA has been isolated from 800 Sacramento suckers from six rivers located in the Sacramento and San Joaquin systems. Eight microsatellite loci have been optimized for use on the MJ-GeneSys BaseStation. Genotypes have been collected for all individuals in five rivers across the eight loci on the MJ-GeneSys BaseStation. A manuscript on the microsatellite development was published recently in the journal Molecular Ecology Notes.

AFLP analyses are complementary to microsatellites because they afford random sampling of a large portion of the genome per unit effort and because no prior sequence data are required for analysis. AFLP screening of all 12 populations has been completed in the past year. The data currently are being analyzed by using a variety of population genetic software packages. Preliminary estimates of average heterozygosity, other metrics of genetic diversity, and population-related measures have been generated using the Arlequin 2.0 software package. Preliminary data indicate that all populations are distinct from one another, average heterozygosity is higher in downstream populations compared to their upstream counterparts, and all upstream populations are most closely related to their downstream counterpart. Genetic patterns among populations are being compared in more detail. The NTSYS-PC software package was purchased recently and is being used to examine patterns of genetic similarity among populations through neighbor-joining and dendrogram methods. Bootstrap values will be generated to test the robustness of these methods.

Population genetic laboratory analyses are nearly complete; and data analyses are underway. Associations between pesticide exposure and variations in population genetic patterns will be tested. Determining whether pesticide exposure influences patterns of genetic variation at a landscape scale also will be tested.

Objective 1—Short-Term Biomarkers and Exposure Analysis

In January and February 2001, a second season of field caging experiments was performed to test whether pesticide exposures are reflected in biomarker responses in the Sacramento sucker. Experiments were timed to coincide with the first rainstorm event after dormant-season application of organophosphate (OP) pesticides to orchards in the Central Valley of California. Field caging sites included the mainstream San Joaquin River downstream of all tributaries (SJ, downstream of agriculture), Orestimba Creek just above the confluence with the San Joaquin (OD, downstream of agriculture), and Orestimba Creek upstream of all agriculture (OU, reference site). Suckers were placed in cages (eight fish per cage), multiple cages were deployed per site, and individual cages were retrieved at different timepoints to evaluate changes in biomarker responses following the rise and fall of pesticide concentrations. Fish were sacrificed, and multiple tissues were excised and archived for subsequent biomarker analysis. DNA strand breaks (comet assay) and acetylcholinesterase activity were analyzed. Concurrently, pesticide concentrations were measured at several timepoints throughout the storm. Finally, to complement DNA strand break data, the Ames mutagenicity assay was included as another indicator of genotoxicity.

Pesticide concentrations were lower than had been observed in previous years at OD. Yet, concentrations of some chemicals were higher in the San Joaquin River. At OD, combined concentrations of diazinon plus methidathion did not exceed 24 ng/L. The reference site at upstream Orestimba Creek did not have detectable concentrations of any of the pesticides. The San Joaquin River site had maximum concentrations of 137 ng/L diazinon and 179 ng/L methidathion, almost twice as high as in 2000, and the entire peak lasted 5 to 6 days. The list of pesticide analytes was expanded from experiments in 2000, and several pesticides other than OPs were detected in the samples, including carbaryl, hexazinone, simazine, molinate, trifluralin, 4-keto-molinate, atrazine, prometryn, metolachlor, thiobencarb, dacthal, pendimethalin, napropamide, and oxyfluorfen.

Biomarker data indicate that San Joaquin River water is genotoxic to test organisms during dormant spray runoff events for the second year in a row. Comet assay results from the field caging experiment indicate background levels of DNA strand breaks in blood from suckers caged early in January, before any runoff events, at the San Joaquin River site compared to controls. A second set of cages were deployed at SJ at the beginning of the main runoff event, and DNA strand breaks were significantly elevated in suckers retrieved at all subsequent timepoints (37-57 percent DNA in comet tail) compared to controls (7-13 percent DNA in comet tail). A third set of cages were deployed after the predicted peak in OP pesticide concentrations. DNA strand breaks were significantly elevated in these suckers as well (57 percent DNA in comet tail) compared to controls (32 percent DNA in comet tail). These results suggest that although induction of DNA strand breaks in fish caged at SJ does not correlate with OP pesticide concentrations, the timing of this biomarker response is correlated with the onset of the pesticide pulse. DNA strand breaks could not be measured in fish caged at OD due to an accident in sample preservation. No significant inhibition of acetycholinesterase enzyme activity was observed at any of the timepoints in either brain or muscle at OD or SJ compared to controls.

Ames mutagenicity assays were conducted on water collected from the San Joaquin effort. This work was conducted in collaboration with Dr. Sergei Kotolevstev of Moscow State University. The expenses for his visit were donated to the project by the Distinguished Research Fellow Program at the Bodega Marine Laboratory. The studies indicated that water collected from the San Joaquin River was mutagenic. These findings, coupled with DNA strand break data, provide strong evidence for the genotoxicity of San Joaquin River water during dormant season runoff events. The Ames assay measures the mutagenic potential of a chemical as its ability to mutate a genetically engineered histidine-requiring strain of Salmonella (S.) typhimurium back to its wild-type phenotype. The engineered strain lacks the ability to grow on agar medium lacking histidine. Mutated bacteria recover the ability to grow on medium lacking histidine, and the mutagenicity of the chemical is measured as the number of treated colonies that grow (number of colonies that have mutated back to the wild-type phenotype) compared to the background mutation rate (indicated by the number of colonies that grow when treated with the dimethylsulfoxide blank). Two strains of S. typhimurium were utilized: TA 98 and TA 100, which are engineered to be sensitive to mutagens that cause frameshift mutations and base substitution mutations, respectively. Two additional manipulations of San Joaquin River water indicate that metals are not responsible for observed mutagenicity indicated by the Ames assay. Metals were removed from San Joaquin River whole water by ion exchange, and this treatment did not reduce mutagenicity in the TA 98 strain. Reconstitution of metals by elution off the ion exchange column into clean water did not restore any mutagenicity.

The finding that San Joaquin River water may be potently genotoxic was presented at the State of the Estuary Conference this year. Key aspects of the work are being repeated, and data are being peer reviewed before submittal for publication. Depending on the outcome of these two efforts, these data could become part of controversial new assessments of the San Joaquin River. Findings could eventually impact the total maximum daily load (TMDL) process for this river system as well as many regulatory activities related to preservation of native fish and to the population status of endangered species such as Chinook salmon. A similar study is being conducted with CALFED, using Chinook salmon smolt caged in the San Joaquin River and a Delta site. Investigators have been in contact with another CALFED-funded study to perform toxicity identification evaluations on the San Joaquin River. Dr. Anderson is collaborating closely with EPA Region 9 scientist, Dr. Bruce Herbold, following a briefing she conducted at EPA last year. It is anticipated that the short-term biomarkers that are being used are valuable indicators of agricultural chemical impacts, and a goal is to dispel the notion that biomarkers are too complicated to actually inform regulatory activities. It is too early to conclude whether the markers of population genetic effect are valuable in detecting impacts of agricultural chemicals. Any conclusion will be reported next year. However, a review of field studies in aquatic organisms that have used population genetic techniques to discern pollution-related effects was published recently, which is a valuable deliverable for this project.

Future Activities:

Genotypes will be collected for the individuals in the remaining river across the eight loci, and data will be analyzed using population genetic software packages. Microsatellite screening of populations should be completed in approximately 2 months, followed by statistical evaluations and population genetic interpretation. AFLP statistical evaluations and interpretation will be completed in approximately 4 months, and a manuscript will be in preparation during the summer. Experiments planned for January and February 2002, will seek to further elucidate toxic substance(s) responsible for genotoxicity in the San Joaquin River.

Experiments planned for winter 2001/2002, include additional laboratory exposure studies. Suckers will be exposed to a cocktail of pesticides, created to mimic chemicals detected in the San Joaquin River during the past 2 years, to test whether detected pesticides alone are capable of inducing biomarker responses observed in 2000/2001 field and laboratory exposure experiments. Furthermore, laboratory exposure to field-collected water experiments will be repeated in the upcoming season, and the Ames assay will be utilized to test various fractions of San Joaquin River water to further elucidate which class(es) of chemical agents may be responsible for previously observed genotoxicity.


Journal Articles on this Report : 3 Displayed | Download in RIS Format

Other project views: All 32 publications 5 publications in selected types All 4 journal articles
Type Citation Project Document Sources
Journal Article Bagley MJ, Anderson SL, May B. Choice of methodology for assessing genetic impacts of environmental stressors: Polymorphism and reproducibility of RAPD and AFLP fingerprints. Ecotoxicology 2001;10(4):239-244. R826603 (2000)
R826603 (2001)
R826603 (Final)
  • Abstract from PubMed
  • Abstract: SpringerLink Abstract
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  • Other: SpringerLink PDF
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  • Journal Article Belfiore NM, Anderson SL. Effects of contaminants on genetic patterns in aquatic organisms: a review. Mutation Research-Reviews in Mutation Research 2001;489(2-3):97-122. R826603 (1999)
    R826603 (2000)
    R826603 (2001)
    R826603 (Final)
  • Abstract from PubMed
  • Abstract: Science Direct Abstract
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  • Other: Science Direct PDF
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  • Journal Article Tranah GJ, Agresti JJ, May B. New microsatellite loci for suckers (Catostomidae): primer homology in Catostomus, Chasmistes, and Deltistes. Molecular Ecology Notes 2001;1(1-2):55-60. R826603 (2001)
  • Abstract: Blackwell-Synergy Abstract
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  • Other: Genome PDF
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  • Supplemental Keywords:

    watersheds, estuary, precipitation, mutagen, ecological effects, population, enzymes, genetic polymorphisms, toxics, ecosystem indicators, aquatic, integrated assessment, environmental chemistry, ecology, genetics, zoology, EMAP, monitoring, western, EPA Region 9, agriculture., RFA, Scientific Discipline, Toxics, Geographic Area, Water, Ecosystem Protection/Environmental Exposure & Risk, Hydrology, Ecology, Water & Watershed, exploratory research environmental biology, Genetics, Environmental Chemistry, Ecosystem/Assessment/Indicators, Ecosystem Protection, Chemistry, pesticides, State, Ecological Effects - Environmental Exposure & Risk, EPA Region, Watersheds, Ecological Indicators, anthropogenic stresses, genotype, pesticide exposure, risk assessment, Region 9, ecological exposure, agricultural watershed, biomonitoring, AFLP, aquatic ecosystems, DNA, pesticide runoff, water quality, fish , California (CA), agriculture ecosystems

    Relevant Websites:

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    Progress and Final Reports:

    Original Abstract
  • 1999 Progress Report
  • 2000 Progress Report
  • 2002
  • Final Report