200 Environmental Toxicology and Chemistry, Vol. 23, No. 1, pp. 200–207, 2004  2004 SETAC Printed in the USA 0730-7268/04 $12.00 + .00 COPPER TOLERANCE IN FATHEAD MINNOWS: I. THE ROLE OF GENETIC AND NONGENETIC FACTORS ALAN S. KOLOK,*² E LIZABETH B. PEAKE,² L AURA L. TIERNEY,² S HAUN A. ROARK,‡ ROBERT B. NOBLE,§ KYOUNGAH SEE,§ and SHELDON I. GUTTMAN‡ ²Department of Biology, University of Nebraska at Omaha, Omaha, Nebraska 68182–0040, USA ‡Department of Zoology, Miami University, Oxford, Ohio, 45056, USA §Department of Mathematics and Statistics, Miami University, Oxford, Ohio, 45056, USA ( Received 11 December 2002; Accepted 20 June 2003) Abstract—Swim performances of male and female fathead minnows (Pimephales promelas) from three different suppliers were determined before and after an 8- to 9-d exposure to 175 g/L copper (Cu). The reduction in swim performance (delta) due to the Cu exposure varied widely among individual fish, but was surprisingly consistent from one supplier to the next and between males and females. Genetic analysis of the individuals revealed significant correlations between delta and genotypic variation at the glucosephosphate isomerase-1, phosphoglucomutase-1, and lactate dehydrogenase-2 enzyme loci. Based upon delta, the most Cu- resistant fathead minnows were bred together, as were the most Cu-susceptible individuals and two groups of unselected minnows. Larvae produced by each group of adults were subjected to a survival test. The median lethal concentration (LC50) for larvae produced by Cu-resistant adults was significantly greater than the LC50s for the control groups. Surprisingly, the LC50 for the larvae produced by Cu-susceptible adults was also significantly greater than the LC50s for the control groups, but not significantly different from the larvae produced by Cu-resistant parents. While Cu tolerance has a genetic component in fathead minnows, the Cu tolerance of larval fish appears to be influenced by nongenetic as well as genetic factors. Keywords—Sublethal exposure Copper Critical swim speed Allozymic variation Median lethal concentration INTRODUCTION Environmental contaminants influence the genetic com- position of fish populations. For example, fish populations living in environments contaminated with heavy metals had reduced genetic variability and/or distinct genetic structure relative to populations from geographically linked, but un- contaminated, reference sites [1–4]. Laboratory studies cor- roborate the hypothesis that environmental contaminants can influence the genetic composition of a fish population. For example, Schlueter et al. [5,6] found that individuals with certain allozyme genotypes survived significantly longer when exposed to copper (Cu) for 96 h. Similar laboratory results have been found for mosquitofish exposed to Cu and cadmium [7], mercury [8], and arsenic and mercury [9]. All of the laboratory studies mentioned above used animal death as their criterion to separate resistant from susceptible individuals. In contrast to this approach, we have been using reduction in aerobic swim performance (preexposure minus postexposure) to differentiate Cu-susceptible fathead minnows (Pimephales promelas) from Cu-resistant ones [10–12]. Using this approach, we have documented physiological differences among individuals that correlate to their ability to swim fol- lowing a sublethal Cu exposure [12]. The physiological dif- ferences that we have identified may be genetically deter- mined; therefore, the first objective of this study was to de- termine whether there were any significant relationships be- tween reduction in swim performance (after Cu exposure) and genetic variation among individual fathead minnows. If correlations exist between genetic variation and Cu tol- * To whom correspondence may be addressed (akolok@mail.unomaha.edu). erance, then the parents may be able to pass their relative tolerance on to their offspring. With this in mind, the second objective of this study was to determine whether larval survival (when exposed to different Cu concentrations in a 7-d survival test) was related to the relative Cu-tolerance of the parents. MATERIALS AND METHODS Fish maintenance Two-month-old fathead minnows (P. promelas) were ob- tained from three national suppliers: Environmental Consult- ing and Testing (Superior, WI, USA), Aquatic Biosystems(Fort Collins, CO, USA), and Aquatic Research Organisms (Hamp- ton, NH, USA). Each stock population was placed in a separate aquarium of aerated, dechlorinated tap water and allowed to acclimate to laboratory conditions (16:8-h light:dark photo- period, 26°C) for 10 d. Then males and females from each stock were separated and placed in six large holding aquaria for an additional two months until they reached maturity. All fish were fed to satiation daily with high-protein, commercial flake food (Aquatic Ecosystems, Apopka, FL, USA). Over the course of these experiments, the laboratory water quality was evaluated on a weekly basis. At 26.0°C, the water in the lab- oratory had the following characteristics: Alkalinity (expressed as mg/L CaCO 3 ) 100  2 (mean  standard error [SE]); hard- ness (expressed as mg/L CaCO 3 ), 196  4; pH, 8.00  0.05; conductivity, 692.1  20.4 S. All aquaria were cleaned daily, which included a static renewal of one-third of the water in each aquarium. Adults The fish from the three stocks grew at different rates. Fish from the Wisconsin (WI) stock grew fastest while fish from