FLOW CYTOMETRY AS A TOOL FOR DETECTING GENOTOXIC EFFECTS IN AMPHIBIANS BREEDING 
IN SOUTHEASTERN MINNESOTA FARM PONDS

Bart L.Bly1, Dean A. Jobe2, Mark B. Sandheinrich1, Melinda G. Knutson3, Brian R. 
Gray3, Shawn Weick3, 1University of Wisconsin-La Crosse, Department of Biology, 
1725 State Street, La Crosse, WI  54601, 2Microbiology Research Laboratory, 
Gundersen-Lutheran Medical Center, 1836 South Ave, La Crosse, WI  54601, 3USGS, 
Upper Midwest Environmental Sciences Center, 2630 Fanta Reed Rd, La Crosse, WI  
54603.
      
High rates of amphibian malformations have been observed in Minnesota and 
several other states.  Flow cytometry is a laboratory test that measures genetic 
damage based on blood samples and may be useful in evaluating sites with 
amphibian malformations.  We conducted a pilot study of farm ponds in 
southeastern Minnesota to assess the feasibility of using flow cytometry to 
assess genetic damage in native amphibians.  We tested associations between the 
type of agricultural land use surrounding a pond and DNA integrity.  Amphibians 
from four reference (one natural wetland, three non-grazed ponds), and five 
exposed (three grazed ponds, and two agricultural ponds) were examined in the 
field for deformities and blood samples were collected for testing with flow 
cytometry.  We assumed that the exposed ponds had higher inputs of agricultural 
contaminants (fertilizers, pesticides, and animal wastes) than the reference 
ponds.  

We found no significant differences in DNA profiles between the reference and 
exposed ponds.  However, several specimens from three different ponds produced 
aneuploid peaks, which is indicative of DNA damage.  We found low correlation 
between amphibian deformities and the coefficient of variation of DNA; 
deformities were rarely observed and they occurred in both the reference and 
exposed ponds.  We found no evidence that surrounding agricultural land use 
affected amphibian DNA integrity or malformation rates in southeastern 
Minnesota.  We demonstrated that flow cytometry could be used to detect and 
quantify DNA damage in native amphibian communities.  It may be useful for 
evaluating amphibian-breeding sites exposed to contaminants or other potential 
genetic stressors.

Keywords:  amphibian malformations, flow cytometry, DNA, agricultural 
contaminants, genetics