2000 Progress Report: Genetic Diversity in California Native Fish Exposed to PesticidesEPA 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, 1999 through January 31, 2000
Project Amount: $649,003
RFA: Ecological Indicators (1998) RFA Text | Recipients Lists
Research Category: Ecological Indicators/Assessment/Restoration , Ecosystems
The overarching goal of this project is to determine the effect pesticide exposure may have on genetic variation in a California native fish. Our first objective is to examine populations of 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. Our 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 USEPA Estuarine and Marine 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. Our 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.
This year, our progress relates mainly to Objective 1. Laboratory research for Objective 2 was completed last year, and a manuscript on the work has recently been accepted for publication in the journal Ecotoxicology. Studies related to Objective 3 will not be initiated until next year.
Objective 1- Population Genetics: In the last year, we have completed
microsatellite and AFLP method development and implementation. We are now
undertaking the task of analyzing DNA from approximately 1000 fin clips
collected at nine reference and five impacted stations in five watersheds. One
strength of this program (described in detail last year) is the extensive
sampling design which allows for a high-level of field replication and a
sophisticated exposure analysis.
We are using microsatellite loci to assess the genetic structure and to detect new mutations in populations of the Sacramento Sucker (Catastomus occidentalis). 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. 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. We hypothesize that this type of mutation is more affected by extrinsic mutagens than are point mutations.
Microsatellite development was one of the major accomplishments for Year Two. We developed a microsatellite library for the Lost River Sucker (Deltistes luxatus). Seventy-four of 250 allelic clones had sequences suitable for developing primers to amplify the microsatellites. An initial screen revealed that 27 of the 74 primer pairs cross-amplified in the Sacramento Sucker (C. occidentalis) and had polymorphic alleles. These 27 microsatellite loci were more intensely screened, and 12 were selected to be used to analyze the population genetic structure of C. occidentalis. We collected individual genotypes from two populations in the Kings River across 12 microsatellite loci using manual gels. We found that the number of alleles per locus ranged from six to 29 and that allele lengths ranged from 89 to 284 base pairs. We also discovered that several loci had abnormal mutations (i.e., there are single or double base pair changes in a four base pair repeat). We are currently optimizing nine to 12 microsatellite loci for use on the MJ-GeneSys BaseStation.
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. To date, optimization of the AFLP protocol for application to Sacramento suckers has been completed, and screening of populations has begun. Of the 96 PCR primer combinations screened, 16 have been selected for use, based on number of bands, banding patterns, and levels of polymorphism. Each primer combination yields between 10 and 50 scorable loci, of which approximately 25 percent are polymorphic. Early efforts included optimizing the PCR thermalcycler profile for selective amplifications, selecting a molecular ladder for use in gels, and developing allelic size standards for each primer combination for use across gels. Screening of populations began in November, 2000.
Objective 1- Short-term Biomarkers and Exposure Analysis: A major accomplishment for this year is the optimization and standardization of the acetylcholinesterase (AChE) enzyme assay for use with fish tissues. Inhibition of cholinesterases (ChEs) is a biomarker of exposure to organophosphate and organocarbamate esters.
We found that fish AChEs were inhibited by the selective inhibitor BW 284c51 and not by the BuChE selective inhibitor iso-OMPA similar to those from mammals and birds. However, the optimum acetylthiocholine substrate concentration in the assay was about 5 mM, several fold higher than that for mammals and birds. This suggests that assays performed mimicking mammal methods may underestimate cholinesterase activity. We also found that the AChE activity of fish muscle was higher than that for brain tissue.
Once the assay conditions were established, we incubated Sacramento Sucker
with different concentrations of diazinon and established our ability to detect
cholinesterase inhibition after organophosphate exposure. At the lowest dose
(0.005 ppm), diazinon exposure caused a 52 percent and 67 percent decrease in
AChE activity from control values in brain and muscle tissues, respectively. At
the highest sublethal dose (0.05 ppm), diazinon exposure caused 90 percent and
In January and February 2000 we performed experiments that included both field caging and laboratory exposure to field-collected water to test whether pesticide exposures are reflected in biomarker responses in Sacramento sucker. Experiments were timed to coincide with the first rainstorm event after dormant-season application of organophosphate pesticides to orchards in the Central Valley of California. Field caging sites included the mainstream San Joaquin River downstream of all tributaries (downstream of agriculture), Orestimba Creek just above the confluence with the San Joaquin (downstream of agriculture), and Orestimba Creek upstream of all agriculture (reference site). Suckers were placed in cages (8 fish per cage), multiple cages were deployed per site, and individual cages were retrieved at different timepoints in order to evaluate changes in biomarker responses following the rise and fall of pesticide concentrations. Ten-gallon composite water samples were concurrently collected and shipped back to BML for controlled laboratory exposures. These field water samples were collected when pesticide concentrations were highest. Sacramento suckers were exposed to field-collected water in the laboratory for 6 days, with water changes every 48 hours, and water quality monitored daily. Fish from both field caging and laboratory exposure experiments were sacrificed, and multiple tissues 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.
Pesticide concentrations were lower than had been observed in previous years at this site, but variations among timepoints were clearly detected. The downstream site at Orestimba Creek had maximum concentrations of 252 ng/L diazinon and 95 ng/L methidathion. 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 77 ng/L diazinon and 23 ng/L methidathion and the entire peak lasted six to eight days. Several other pesticides were measured but were not detected in our samples.
Comet assay results from the field caging experiment indicate elevated DNA strand breaks in blood from suckers caged at the San Joaquin River site (33 percent to 45 percent of DNA in comet tail) compared to controls (9 percent to 16 percent of DNA in comet tail). These elevations in strand breaks occurred at all timepoints, including before, during, and 6 days after the peak pesticide pulse, with no pattern coinciding with time. There was no difference in DNA strand breakage between fish caged at the Orestimba Creek site downstream of agriculture compared to control fish at all timepoints including during, and 6 days after, the peak pesticide pulse. Inhibition of AChE enzyme activity was observed following the peak of pesticide runoff. However, concentrations of measured compounds were too low to elicit the effects observed.
Comet assay results for the laboratory exposure to field-collected water experiment indicate elevated DNA strand breaks in fish exposed to water collected from the contaminated San Joaquin River site (28 percent of DNA in comet tail), compared to fish exposed to water collected from the control site (9 percent of DNA in comet tail). The degree of DNA damage in fish exposed to San Joaquin River water is comparable between lab and field experiments. However, fish exposed to contaminated Orestimba Creek water had slightly elevated DNA damage following laboratory exposure, but no increase in damage following field exposure. This discrepancy may be explained by the fact that caged fish were exposed to peak pesticide concentrations for a relatively short period of time (less than 10 hours) due to rapid dilution by clean water from the upper watershed, whereas fish in the laboratory were exposed to peak pesticide concentrations for 6 days. Brain acetylcholinesterase enzyme activity was inhibited in the samples with the highest pesticide concentrations, including samples from an additional site, the Feather River.
Experiments planned for winter 2000/2001 include additional laboratory exposure studies using year 2001 environmental chemistry monitoring at field sites to guide our selection of compounds. Furthermore, field caging and laboratory exposure to field-collected water experiments will be repeated in the upcoming season. An attempt will be made to characterize the general class of toxicants eliciting DNA strand breaks in the San Joaquin River, and we will increase the number of pesticides measured. In addition, AFLP and microsatellite analyses will continue throughout the year.
Journal Articles on this Report : 2 Displayed | Download in RIS Format
|Other project views:||All 32 publications||5 publications in selected types||All 4 journal articles|
||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.||
||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.||