DIFFERENTIAL SURVIVAL OF FATHEAD MINNOWS, PIMEPHALES PROMELAS, AS AFFECTED BY COPPER EXPOSURE, PRIOR POPULATION STRESS, AND ALLOZYME GENOTYPES

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1 Environmentl Toxicology nd Chemistry, Vol. 16, No. 5, pp , SETAC Printed in the USA /97 $ DIFFERENTIAL SURVIVAL OF FATHEAD MINNOWS, PIMEPHALES PROMELAS, AS AFFECTED BY COPPER EXPOSURE, PRIOR POPULATION STRESS, AND ALLOZYME GENOTYPES MARK A. SCHLUETER,* SHELDON I. GUTTMAN, JAMES T. ORIS nd A. JOHN BAILER Center for Environmentl Toxicology nd Sttistics, Deprtment of Zoology Center for Environmentl Toxicology nd Sttistics, Deprtment of Mthemtics nd Sttistics, Mimi University, Oxford, Ohio 45056, USA (Received 14 Mrch 1996; Accepted 20 September 1996) Abstrct Two popultions of fthed minnows (F 1, offspring of survivors of n cute fluornthene exposure; N 1, nive htchery fish born in outdoor ponds) were concurrently exposed to pproximtely 850 g/l of copper for 132 h. During the exposure, 49% of the F 1 nd 85% of the N 1 minnows died. A curled operculum deformity, detected in 54% of the F 1 popultion, ws significntly relted to mortlity. A filure time regression model, combining both the F 1 nd N 1 popultions together, ws fit to exmine the reltionship between popultion type (F 1 or N 1 ), body condition (weight/length 3 ), presence of n operculum deformity, nd different llozymes on time to deth (TTD). The model indicted tht type of popultion, body condition, the presence of n operculum deformity, nd three loci (GPI-1*, IDHP-1*, nd MDH-2*) were significntly relted to TTD. The F 1 minnows hd higher survivl rte nd longer verge TTD compred to N 1 minnows. In comprison to the N 1 popultion, the F 1 popultion possessed higher frequency of genotypes ssocited with incresed survivorship t the IDHP-1* nd MDH-2* loci. Weight (nd body condition) ws negtively correlted with survivorship. Minnows with severe operculum deformity, believed to be result of prentl exposure to fluornthene, hd 100% mortlity rte nd exhibited considerbly reduced TTD compred to nondeformed minnows. Multilocus heterozygosity ws not relted to TTD for either popultion. This study indictes tht genetic fctors my exhibit stronger effects on survivorship thn other fctors (e.g., weight/body condition) commonly ssocited with fitness. Keywords Allozyme electrophoresis Accelerted filure time Regression model Weight effects Fluornthene INTRODUCTION Popultions undergo mny chnges to dpt nd survive during exposure to stress. These chnges include short-term physiologicl dpttion nd long-term genetic selection. The importnce of genetic vrition in popultions exposed to environmentl chnge nd/or stressors hs been previously documented [1]. Mny studies hve linked specific genotypes to enhnced pollution tolernce in fish [2 8] nd invertebrtes [8 13]. Most helthy popultions re chrcterized by high levels of genetic diversity [8,14,15]. Popultions exposed to environmentl pollutnts show n incresed level of contminnt tolernce compred to nonexposed popultions; however, they lso exhibit decresed genetic diversity [1,8]. By pplying second stress to previously stressed popultion or geneticlly bottlenecked popultion, informtion on the popultion s bility to resist nd dpt to future chnges/stress cn be obtined. The following study exmined the impct of second stressor (copper) on popultion previously stressed by fluornthene. The study ws prt of lrger project exmining the long-term effects of fluornthene on fish. Fluornthene, phototoxic polycyclic romtic hydrocrbon (PAH) is model compound for the study of PAHs. It is suspect crcinogen nd hs been shown to be mutgenic [16,17]. The primry sources of fluornthene nd other PAHs in qutic environments re from col nd other fossil fuel * To whom correspondence my be ddressed. distilltion nd storge fcilities, exhust from internl combustion engines, nd other combustion sources [18]. Copper, n essentil element, becomes toxic in qutic environments when concentrtions exceed 10 g/l. In qutic nimls, uptke of excess copper occurs t the gills during respirtion. Copper enters qutic environments from mny sources including wstewter from metl processing, pickling, nd plting bths; industril wste (often first given off to the ir); nd from copper sulfte, which is used s n lgecide [19]. The min objective of the present investigtion ws to exmine the survivl of two fthed minnow (Pimephles promels) popultions, one nive (pond rised) popultion (N 1 ) nd one selected popultion (F 1 ), during n cute exposure to copper (previously identified s differentilly ffecting llozyme genotypes [2]). The selected popultion ws the first filil (F 1 ) genertion born from the survivors (P 1 genertion) of n cute exposure to fluornthene [20] (Fig. 1), toxicnt with different mode of ction thn copper. It ws hypothesized tht the F 1 popultion would survive the copper exposure differently thn the N 1 popultion, becuse the P 1 genertion underwent selection event during the fluornthene exposure possibly resulting in their offspring (the F 1 popultion) hving less genetic vrition. A second objective ws to evlute whether llele nd genotype frequencies were ssocited with survivl in the two popultions, nd to compre those dt with previously identified key genotypes [2]. 939

2 940 Environ. Toxicol. Chem. 16, 1997 M.A. Schlueter et l. mortlity (LC90) for the copper exposure. Rnge-finding tests were conducted using six 16-L plstic continers filled with 12 L of dechlorinted wter. Ten minnows were plced in ech continer. Fish were exposed to five concentrtions of copper nd no-copper control. Three concurrent replictes of the rnge-finding experiment were conducted. A probit regression nlysis ws used to estimte n LC90. The three estimtes of the LC90, bsed on three different replictes of the rngefinding experiments, were 950, 800, nd 800 g/l. The men of the three estimtes, 850 g/l, ws used s the trget concentrtion for the copper exposure. Copper tretments were chieved using concentrted stock solution mde from nhydrous copper sulfte (CuSO 4 ) (Fisher Scientific, Cincinnti, OH, USA), nd mintined by dily sttic renewl. Wter chemistry prmeters (ph, temperture, dissolved O 2, hrdness, lklinity, nd conductivity) were mesured t the beginning nd t the completion of the test. Copper concentrtions of ech tretment were mesured dily (see wter chemistry section below). Fig. 1. The fthed minnow popultions nd their origins re illustrted. The originl popultion of minnows exposed to fluornthene nd the N 1 (nive) popultion were directly obtined from the fish htchery ponds. The P 1 popultion ws the survivors of the cute fluornthene exposure nd the F 1 popultion ws the offspring of the P 1 popultion. Experimentl fish METHODS Two different popultions of juvenile fthed minnows were used in this study. The first popultion included 656 F 1 fthed minnows tht were the offspring of the survivors (5% of the originl prentl popultion) of n cute exposure (x SD: 87 5 g/l) to fluornthene [20]. The originl fish for the fluornthene exposure nd 770 nive (N 1 ) fthed minnows comprising the second popultion of this study were obtined from Kurtz s Fish Htchery (Elverson, PA, USA). The verge weight ( stndrd devition) of 656 F 1 fthed minnows ws g with n verge stndrd length of cm. The verge weight of 770 N 1 fish ws g with n verge stndrd length of cm. Fish were mintined nd cclimted for 4 weeks prior to testing in 115-L flow-through tnks provided with 18 C constntly erted dechlorinted wter under 12L:12D photoperiod. They were fed diet of ground Purin Trout Chow (St. Louis, MO, USA) nd Argentemi Grde III brine shrimp (Argent Chemicl Lbortories, Redmond, WA, USA) twice dy. Rnge-finding tests Nive (N 1 ) minnows were used in 96-h rnge-finding experiments to determine trget concentrtion yielding 90% Filure time experiments For the time-to-deth exposure, 606 F 1 fish nd 570 N 1 fish were plced into two different 115-L experimentl tnks nd 50 fish from the F 1 nd N 1 popultions were plced into seprte 58-L control tnks. Fish were cclimted in their tnks for 48 h prior to the exposure. New wter ws dded to ech tnk fours times dily for totl of one volume ddition/d. Overflow wter ws removed vi stndpipe. The trget concentrtion, 850 g/l of copper, ws introduced in the form of nhydrous CuSO 4 with n initil direct dose to the experimentl tnk nd mintined by dosing the renewl wter with nhydrous CuSO 4. The ctul copper concentrtion ws mesured using tomic bsorption spectroscopy t lest once dy t the end of the renewl cycle. Ded fish were removed every 4 h during the exposure. Deth ws determined to hve occurred if fish ws not ventilting nd if no operculum movement ws pprent for 30 s. Ech ded fish ws then weighed, mesured for stndrd length, nd plced in plstic tube for subsequent electrophoretic nlysis. The plstic tubes were held briefly ( 1 h) on ice, nd then trnsported nd stored in 70 C freezer. The exposure ws terminted t 132 h, becuse t tht time no mortlity occurred during the previous 18-h period in either popultion. At the end of the 132-h exposure, fish still live in the experimentl tnks were scrificed, weighed, mesured for stndrd length, plced in plstic tube, nd stored t 70 C. Wter chemistry Wter chemistry prmeters were recorded every 24 h from the beginning of the 132-h exposure. Dissolved oxygen nd temperture were mesured directly in the tnks with Model 51B dissolved oxygen meter (Yellow Springs Instrument Compny, Yellow Springs, OH, USA). A wter smple ws tken for ph, which ws mesured immeditely. Additionl wter smples were tken nd stored t 4 C until nlyzed the next dy for hrdness, lklinity, nd conductivity. Wter smples used for copper nlysis were cidified to 1% with nitric cid nd stored t 4 C for 1 to 2 weeks until nlysis. An Orion Ionnlyzer 901 equipped with n Orion 8130 Ross combintion electrode (Orion Reserch, Cmbridge, MA, USA) ws used to mesure ph. Conductivity ws mesured with Model 2510 Conductivity Meter (Hch Chemicl Com-

3 Differentil survivl of fthed minnows Environ. Toxicol. Chem. 16, buffer nd tris citrte (ph 6.3) electrode buffer [24]. Gels were stined by stndrd methods [24,25]. Isozyme loci were numbered bsed on their migrtion distnce from the origin; the locus with the gretest migrtion ws lbeled one, the locus with the second lrgest migrtion ws lbeled two, nd so on. Alleles were lbeled in similr mnner. The llele with the gretest migrtion distnce ws lbeled, the one with the next lrgest migrtion distnce ws lbeled b, nd so on. Fig. 2. Differentil survivorship of F 1 nd N 1 (nive) juvenile fthed minnows cutely exposed to copper for 132 h. The F 1 popultion ws the offspring of survivors of n cute fluornthene exposure. pny, Des Moines, IA, USA). Totl lklinity ws determined using n lklinity test kit (Hch Chemicl Compny). Hrdness ws mesured by stndrd titrtion method [21]. Copper concentrtions were determined using Vrin SpectrAA- 10/20 furnce tomic bsorption spectrophotometer (Vrin Instruments, Inc., Sugr Lnd, TX, USA). The stndrd methods suggested by Vrin were used for copper mesurement [22]. Concentrtions of the copper stndrds nd smples were detected by mesuring bsorbnce t nm wvelength with 0.5 nm slit width. The shing temperture used ws 675 C, nd the tomiztion temperture ws 2,300 C. Genetics (electrophoresis) The minnows were ground in sucrose 2% 2-phenoxyethnol solution t rtio of 1:1 (v/v) nd then centrifuged in their plstic tubes t 8,160 g for 20 s. The superntnt ws bsorbed onto filter pper wicks tht were loded into 15% strch gels (hydrolyzed potto strch; Sigm Chemicl Compny, St. Louis, MO, USA). Seven vrible gene loci from five enzyme systems (bbrevition nd E.C. numbers in prentheses) were exmined: bet glctosidse ( -GAL*, ), glucose-6-phosphte isomerse (GPI-1*, GPI-2*, ), isocitrte dehydrogense (IDHP-1*, IDHP-2*, ), mlte dehydrogense (MDH-2*, ), nd phosphoglucomutse (PGM*, ) [23]. The -GAL*, IDHP*, nd MDH* were run on tris citrte (ph 8.0) buffer, nd GPI* nd PGM* were run on tris citrte (ph 6.7) gel Dt nlysis Attributes of the electrophoretic dt including llele nd genotype frequencies, men heterozygosity, nd goodness of fit of the dt to expected Hrdy Weinberg equilibrium proportions were nlyzed using chi-squre goodness-of-fit tests s implemented in the computer progrm BIOSYS-1 [26]. Accelerted filure time regression models were creted using the time to deth (TTD) dt nd the survivl time nlysis procedures in SAS version 6.03 LIFEREG routine [27]. The model describes the reltionship between filure time (TTD), nd severl predictor vribles/covrites [28]. The functionl form of this model is where log(t i ) f(x i ) e i t i TTD or time of censoring of individul i x i vector of p predictor vribles (x i1, x i2,...,x ip ) f(x i ) liner combintion of covrites ( 0 1 x i1 2 x i2... p x ip ) scle prmeter e i error term for individul i Thus, j (where j 1,..., p) is prmeter tht mesures the chnge in log TTD s covrite j is incresed by one unit holding other covrites constnt. If j is positive (negtive), then increses in the covrite re ssocited with increses (decreses) in survivl time. Certin predictor vribles such s the presence of operculum deformity were ctegoricl. These vribles were entered into the regression model s series of indictor vribles. As n exmple, operculum deformity ws two-level ctegoricl vrible present or bsent. An indictor vrible for operculum deformity, sy X, ws defined s X 1 if the operculum deformity ws present nd X 0 if not. To select the error distribution, the survivl dt were evluted grphiclly. As n exmple, if the dt followed Weibull distribution, the plot of log[ log(proportion live)] versus log time, Tble 1. Mens (stndrd devitions) of wter chemistry prmeters for the F 1 nd N 1 copper exposures nd their controls Prmeters F 1 F 1 control N 1 N 1 control ph 8.39 (0.06) 8.32 (0.08) 8.32 (0.05) 8.42 (0.11) Temperture ( C) 24.0 (0.0) b 24.0 (0.0) b 24.0 (0.0) b 24.0 (0.0) b Dissolved O 2 (mg/l) 6.76 (0.29) 6.87 (0.35) 6.67 (0.37) 6.68 (0.27) Conductivity ( ohms) 904 (23) 897 (69) 907 (69) 925 (38) Hrdness ( g/l CCO 3 ) 346 (8) 330 (9) 346 (7) 338 (8) Alklinity (mg/l) 260 (4) 262 (7) 261 (5) 264 (6) Copper ( g/l) 847 (31) 5 c 852 (16) 5 c F 1 offspring of survivors of n cute fluornthene exposure, N 1 nive htchery fish. b No devition bsed on the detectbility of the instrument. c Copper concentrtions in the control tnk were below the detection limit of the tomic bsorption spectrophotometer.

4 942 Environ. Toxicol. Chem. 16, 1997 M.A. Schlueter et l. Tble 2. Test of Hrdy Weinberg equilibrium using chi-squre nlysis of genotype frequencies for F 1 fthed minnows exposed to copper. A p vlue 0.05 indictes tht the locus does not devite significntly from Hrdy Weinberg equilibrium Locus Genotype Observed Expected 2 Vlue p Vlue -GAL* b c bb bc cc GPI-1* b c bb bc cc b GPI-2* b c bb bc cc b IDHP-1* b bb IDHP-2* b bb MDH-2* b c d bb bc bd cc cd dd c PGM* b c bb bc cc F 1 offspring of survivors of n cute fluornthene exposure. b Chi-squre test pooling rre genotypes yielded p vlue c Chi-squre test pooling rre genotypes yielded p vlue (indicting the locus ws in Hrdy Weinberg equilibrium). would be pproximtely liner [29]. A grphicl nlysis of the TTD results suggested tht Weibull distribution ws dequte, nd thus this prmetric form ws used in ll subsequent nlyses. For more detiled explntion of sttisticl terms nd nlysis, see Dixon nd Newmn [28]. RESULTS Exposure A totl of 298 (49%) F 1 nd 484 (85%) N 1 fthed minnows died during the 132-h copper exposure in the exposure tnks, wheres no fish died in the control tnks. Mortlity curves for the copper exposures re presented in Figure 2. Wter qulity prmeters were similr for both the exposure nd control tnks (Tble 1). Deformity An operculum deformity ws detected in 54% of the F 1 popultion [20]. The deformity consisted of n nterior curling Tble 3. Test of Hrdy Weinberg equilibrium using chi-squre nlysis of genotype frequencies for N 1 (nive htchery) fthed minnows exposed to copper. A p vlue 0.05 indictes tht the locus does not devite significntly from Hrdy Weinberg equilibrium Locus Genotype Observed Expected 2 Vlue p Vlue -GAL* b c bb bc cc GPI-1* b c bb bc cc GPI-2* b c bb bc cc IDHP-1* 40 b 177 bb IDHP-2* bb MDH-2* c cc PGM* b c d bb bc bd cc cd dd of the operculr skin flps locted long the cudl mrgin of the bony opercul, directly exposing the gills to the wter. The deformity ws judged to be severe in 4% of this popultion. The operculr deformity present in the F 1 popultion ws ttributed to the cute fluornthene exposure of the P 1 genertion [20]. Deformed fish hd much lower weight (nd body condition) thn norml fish. The severely deformed fish hd n verge weight of g (n 23) compred to the verge weight of g (n 583) for norml fish. Genetics The observed genotype frequencies, expected genotypic proportions, nd goodness of fit for ech enzyme locus re presented in Tbles 2 nd 3. In the F 1 popultion, the MDH- 2*, GPI-1*, nd GPI-2* loci devited significntly from Hrdy Weinberg equilibrium. When rre genotypes were pooled, MDH-2* response ptterns did not differ from equilibrium, becuse the disequilibrium ws cused by single *bc individul. The GPI-2* locus ws not in equilibrium due to n unusully high number of rre homozygous *cc genotypes. In the N 1 popultion the MDH-2* locus ws not in equilibrium becuse of low number of *c heterozygotes. Models In the present study, two ccelerted filure time regression models were creted. The first model incorported the follow-

5 Differentil survivl of fthed minnows Environ. Toxicol. Chem. 16, Tble 4. Accelerted filure time regression model 1: summry tble of locus effects on time to deth (TTD) for both F 1 nd N 1 popultions of fthed minnows exposed to copper (d.f. degrees of freedom, ˆ estimte of covrite effect on survivl time; log TTD/g, log TTD/group, or log TTD/genotype) Vrible Prmeter d.f. ˆ (SE) 2 Vlue p Vlue Intercept Intercept (1.291) Condition Overll (0.276) Deformity Overll No (0.518) Yes * (0.000) -GAL* Overll b (1.554) bb (0.327) bc (0.000) GPI-1* Overll b (0.581) c (0.736) bb (0.540) bc (0.565) cc * (0.000) GPI-2* Overll (1.183) b (0.558) bb (0.521) bc (0.522) cc * (0.000) IDHP-1* Overll (0.253) b (0.133) bb * (0.000) IDHP-2* Overll b (0.589) bb * (0.000) MDH-2* Overll (1.106) c (0.895) cc (0.875) cd * (0.000) PGM* Overll (0.218) b (0.381) c (0.137) bc (0.050) cc * (0.000) Popultion Overll F (0.050) N 1 * (0.000) Scle Overll (0.050) F 1 offspring of survivors of n cute fluornthene exposure, N 1 nive htchery fish. * reference genotype for ech locus. ing predictor vribles: fish popultion (F 1 or N 1 ), body condition (weight/length 3 ), presence of operculum deformity, nd genotypes t seven vrible loci (Tble 4). The second model, which exmined the effect of heterozygosity in F 1 nd N 1 fish, incorported the following predictor vribles: body condition, presence of operculum deformity, nd number of heterozygous loci (Tble 5). Body condition ws found to significntly ffect TTD (p ) for both F 1 nd N 1 minnows in ll models. Ech model ssocited body condition with negtive, indicting tht fish with lower body condition hd longer predicted TTD thn did fish with higher body condition. Becuse these fish were ll of the sme reltive length, body condition ws highly ssocited with fish weight. In fct, fish weight ws inserted in plce of body condition in both models nd yielded the sme significnt (p ) result. There ws not significnt ssocition between length nd TTD. In the F 1 popultion, TTD ws decresed by the presence of the operculum deformity ( , p 0.03). Genotypes of three of the seven mesured vrible loci were significntly relted to TTD (Tble 4). The loci GPI-1* ( , p 0.02), IDHP-1* ( , p ), nd MDH-2* ( , p ) were strongly relted to TTD. Overll heterozygosity, the sum of the heterozygous loci present, rnging from zero to four loci in ech individul, ws not significntly ( , p 0.38) relted to TTD in either the F 1 or the N 1 fish (Tble 5). A logistic regression for different survivl weight reltionships ws fit for genotypes (in the IDHP-1 locus) exhibiting differentil survivl. This model confirmed our ssertion tht both genotype nd weight were significnt fctors in predicting survivl (nlysis not reported). Differences in size clsses The F 1 nd N 1 fthed minnow popultions were ech seprted into three size clsses, with pproximtely the sme number of fish in ech clss, to exmine the negtive reltionship between size nd TTD nd to determine if ny loci were correlted with these size differences. Consistent with the previous filure time regression nlysis, the three size clsses in both the F 1 nd N 1 popultions showed significntly different (p ) survivorship ptterns, bsed on chisqure tests. In the F 1 popultion, 69% of the lrge fish ( g), 46% of the medium fish ( g), nd 34% of the smll fish ( g) died during the exposure (Fig. 3). For the N 1 popultion, 97% of the lrge ( g), 93% of the medium ( g), nd 63% of the smll fish ( g) died during the exposure (Fig. 3b). It should be noted tht the smll size clss ( g) of the N 1 popultion ws pproximtely equl to both the medium nd smll size clsses of the F 1 popultion. This my explin the response of the N 1 medium size clss, which exhibited survivorship response just slightly better thn the lrger size clss, rther thn the predicted intermedite response (Fig. 3b). Significnt frequency differences in the genotypes t the IDHP-1* ( , p ) nd the MDH-2* ( , p 0.04) loci, both of which were ssocited with TTD nd weight (Tble 4), were detected between the F 1 nd N 1 popultions. At the IDHP-1* locus, survivorship decresed wheres weight incresed from genotypes *bb *b *. For the MDH-2* locus, survivorship decresed wheres weight incresed from genotypes *cc *c *. The IDHP-1* bb nd the MDH-2*cc genotypes hd the lowest verge weight (Tble 6), while exhibiting n incresed frequency in the surviving minnows compred to ded minnows in both the F 1 nd N 1 popultions. This reltionship between size nd certin genotypes resulted in smller (reltive to weight or body condition) fish surviving longer (greter TTD) thn lrger fish. The verge weight for fish with both the IDHP-1*bb nd the MDH-2*cc genotypes ws g (F 1 popultion, n 270) nd g (N 1 popultion, n 232) compred to the verge weight of g (F 1, n 336) nd g (N 1, n 338) for fish with ll other genotype combintions. Fthed minnows tht possessed both the resistnt MDH-

6 944 Environ. Toxicol. Chem. 16, 1997 M.A. Schlueter et l. Tble 5. Accelerted filure time regression model 2: summry tble of multiple locus heterozygosity effects on time to deth (TTD) for F 1 nd N 1 fthed minnows exposed to copper (d.f. degrees of freedom. ˆ estimte of covrite effect on survivl time; log TTD/g or log TTD/genotype) Vrible Prmeter d.f. ˆ (SE) 2 Vlue p Vlue Intercept Intercept (0.616) Condition Overll (0.080) Deformity Overll No (0.447) Yes (0.000) Heterozygosity (no. of loci) Overll (0.536) (0.530) (0.532) (0.561) * (0.000) Popultion Overll F (0.182) N (0.000) Scle Overll (0.048) F 1 offspring of survivors of n cute fluornthene exposure, N 1 nive htchery fish. * reference genotype for ech locus. 2*cc nd IDHP-1*bb genotypes survived longer thn minnows tht hd either one or neither of these resistnt genotypes. In the F 1 popultion 56.7% tht possessed both the resistnt MDH-2*cc nd IDHP-1*bb genotypes survived the exposure, compred to 26.6% survivorship rte for F 1 minnows with ny other genotype combintion. A similr response ws found Fig. 3. Differentil survivl of the three size clsses for the F 1 () nd N 1 (nive) popultions (b). Smll minnows were found to exhibit significntly better survivorship (p ) thn the medium or lrge fish in both popultions. The F 1 popultion ws the offspring of the survivors of n cute fluornthene exposure. in the N 1 popultion, where minnows with both resistnt genotypes survived considerbly better thn minnows with ny other genotype combintion (Fig. 4). A significntly greter frequency of individuls with the copper resistnt *bb genotype t the IDHP-1* locus ws present in the F 1 popultion compred to the N 1 popultion. Tble 7 illustrtes the overll increse in IDHP-1*b llele frequency from the fluornthene-exposed popultion [20], to the exposure s survivors (the F 1 s prents), nd to the F 1 popultion. DISCUSSION Single locus nd body condition effects on TTD Three enzyme loci (GPI-1*, IDHP-1*, MDH-2*), fish condition (weight), fish popultion, nd presence of n operculum deformity were significntly relted to TTD during the 132-h copper exposure. A negtive reltionship between fish condition nd TTD ws detected during the cute copper exposures. Smller fish (reltive to weight or condition) showed higher survivorship thn medium fish, which survived better thn lrge fish. This study showed tht incresed survivorship of fthed minnows ws possibly ssocited with the IDHP- 1*b nd the MDH-2*c lleles. Individuls homozygous for the IDHP-1*b nd the MDH-2*c lleles hd the highest survivorship, wheres heterozygous individuls possessing these lleles showed moderte survivorship, nd homozygous individuls possessing other lleles hd the poorest survivorship (Tble 6). These genotypes were lso significntly relted to decresed weight ( 0 in the filure time regression models); verge weights of survivors of the exposure were less thn those of ded fish. Similr negtive reltionships between weight, genotype, nd TTD hve been previously documented [2]. The tested loci (e.g., IDHP, MDH) my be linked to genes tht re involved in the stress response or re relted to physicl prmeter (e.g., size); these ssocitions between physicl prmeters nd/or stress response my lso explin the incresed survivorship in certin fish with genotypes of tested loci. Smller fish might lso hve been more tolernt due to their higher metbolic rte compred with lrger fish, which my hve enbled them to more effectively meet the incresed mintennce demnds of the copper exposure [30 32].

7 Differentil survivl of fthed minnows Environ. Toxicol. Chem. 16, Tble 6. Genotype frequencies for the IDHP-1* nd the MDH-2* loci re listed for the F 1 nd N 1 popultions. Sttisticl nlyses indicted significnt difference in llele frequencies cross the different weight clsses for the IDHP-1* (p ) nd the MDH-2* (p ) loci for both popultions. Differences in genotype frequency within groups were tested; results re listed below the genotype frequencies (NS not significnt, * significnt [p 0.05], nd ** highly significnt [p 0.001]). The IDHP-1*bb nd the MDH-2*cc genotypes were significntly ssocited with incresed survivorship. Individuls were grouped into three size clsses of pproximtely the sme size; due to the poorer resolution of the IDHP-1* locus, there were fewer dt points compred to the MDH-2* locus for ech size clss Genotypes Ded b Smll fish Alive Ded Medium fish Alive Ded Lrge fish Alive Ded Totl fish Alive Averge weight (g) (SE) IDHP-1* F1 popultion (n 58) (n 121) (n 84) (n 108) (n 108) (n 48) (n 250) (n 277) * (0.005) *b (0.000) *bb (0.000) (NS) (**) (**) (**) MDH-2* (n 69) (n 132) (n 95) (n 111) (n 131) (n 64) (n 295) (n 307) *c (0.002) *cc (0.000) *cd (0.008) (*) (**) (**) (**) N1 popultion IDHP-1* (n 60) (n 108) (n 83) (n 62) (n 83) (n 57) (n 226) (n 227) * (0.006) *b (0.000) *bb (0.008) (**) (NS) (NS) (*) MDH-2* (n 80) (n 108) (n 113) (n 69) (n 132) (n 68) (n 325) (n 245) * (0.001) *c (0.002) *cc (0.000) (NS) (NS) (NS) (*) F 1 offspring of survivors of n cute fluornthene exposure, N 1 nive htchery fish. b LT50 medin lethl time. Differences in F 1 nd N 1 survivl The first plusible reson for the significnt difference in survivorship existing between the F 1 nd N 1 popultions my hve been due to n incresed frequency of resistnt *bb genotypes t the IDHP-1* locus in the F 1 fish. This increse my be the result of selection. Selection is process tht results in chnge in reltive frequency of genotypes due to differences in the bility of their phenotypes (e.g., llozymes) to chieve representtion in the next genertion. This occurs becuse individuls with certin phenotypes (e.g., llozymes) exhibit differentil fitness under vrious conditions or stresses (e.g., predtor resistnce, dpttion to chnges in hbitt, toxicnt exposure). Acute exposure of the P 1 popultion my hve led to n incresed frequency of the *bb genotype (or llele *b) t the IDHP-1* locus resulting in incresed survivorship for the F 1 popultion, becuse this genotype ws linked with copper resistnce. Tble 7 illustrtes the increse of the *b llele cross the popultions (fluornthene exposed popultion fluornthene survivors [P 1 ] F 1 popultion). However, if prior selection hd decresed or deleted the *bb genotype (or the *b llele), the F 1 popultion should hve hd poorer survivorship. In this scenrio, popultion is exposed to stress (e.g., fluornthene), which selects for specific genotypes (deleting other genotypes in the process). Subsequently, second stress (e.g., copper) selects ginst the remining genotypes, resulting in decrese or even extinction of the popultion/species. Helthy popultions generlly hve lrge mount of genetic vrition, which increses the probbility tht they possess tolernt genotypes nd cn dpt to chnges nd stresses in their environment [1,2]. In seprte study, F 1 nd nive fish were cutely exposed to fluornthene [20]. The F 1 fish exhibited much poorer sur- Fig. 4. The frequency of surviving fthed minnows cutely exposed to copper with both the MDH-2*cc nd IDHP-1*bb genotypes compred with ll other genotype combintions. A significnt difference in survivorship between genotype combintions ws observed in both the F 1 nd N 1 (nive) survivors. An sterisk (*) denotes sttisticlly significnt (p 0.05) difference between the ctegories in ech popultion. The F 1 popultion ws the offspring of the survivors of n cute fluornthene exposure.

8 946 Environ. Toxicol. Chem. 16, 1997 M.A. Schlueter et l. Tble 7. Frequency of the IDHP-1* lleles in the popultion exposed to fluornthene, the fluornthene exposure survivors (P 1 ), the offspring of the survivors of the fluornthene exposure (F 1 ), nd the nive (N 1 ) popultion. Stndrd devitions re reported in prentheses. A significnt (p 0.001) increse in the resistnt IDHP-1*b llele ws seen from the fluornthene-exposed popultion to the F 1 popultion Allele Fluorntheneexposed popultion Fluornthene survivors (P 1 ) F 1 popultion N 1 popultion * (0.010) (0.028) (0.010) (0.014) *b (0.010) (0.028) (0.010) (0.014) vivl thn did the nive fish, suggesting tht fluornthene tolernce ws not ttined in the F 1 popultion. The investigtors ttributed the decresed fluornthene tolernce in the F 1 fish to the curled operculum deformity. The operculum deformity resulted in the direct exposure of the gills to ultrviolet (UV) light, which enhnced the effects of the phototoxic fluornthene. A second plusible explntion for incresed survivorship of the F 1 popultion my be tht the fluornthene exposure selected minnows tht were slow growers. Kolok nd Oris [33] found tht within popultion of fthed minnows there were fst nd slow growers. Rpid growth, lthough significnt reproductive dvntge, my be detrimentl to overll physicl condition. Fthed minnows tht grew fst exhibited poorer swimming performnce, possibly indicting reduced level of fitness [33]. This growth pttern is possible dpttion to the unpredictble wether ptterns during the breeding seson. Fst growers would mture nd breed erly in the breeding seson, wheres the slow growers would mture nd breed lte in the breeding seson. In effect, the breeding of the entire popultion would be spred out, incresing the probbility tht portion of the lrve would htch during n optiml time of the seson. Bsed on this concept of slow nd fst growers, it is possible tht the fluornthene exposure selected for the slow growers. These fish my hve been more resistnt to the toxicnt (copper nd/or fluornthene) stress, due to their different metbolic rte. This might explin why the verge weight (or body condition) of the survivors ws much less thn tht of the ded minnows nd why the frequency of IDHP-1* llele *b incresed over tretment nd time (Tble 7), becuse this llele ws ssocited with low fish weight. Operculum effects on TTD The mjority of deformed fish died in the first 18 h of the exposure. Incresed mortlity from the operculum deformity ws possibly due to the disruption of the negtive pressure ventiltion used by ll members of the clss Osteichthyes [34]. Becuse the deformity disrupts the cretion of negtive pressure, operculr efficiency is compromised. Rpid mortlity in the operculr deformed fish during the copper exposure ws possibly the result of providing dditionl stress (e.g., copper) on n lredy stressed (e.g., poor ventiltion) nd wekened group of minnows. Heterozygosity effects Heterozygosity did not ffect TTD. Similr results hve been documented in other studies [2,7,35], which found tht TTD ws dependent on llelic genotype, but not heterozygosity. If n llele t n individul locus (e.g., IDHP-1*) strongly ffects survivorship, then being homozygote t tht locus my be more significnt to n individul s fitness thn overll heterozygosity [2]. Conclusions The results of the present study were different thn wht current hypotheses suggest. A selected popultion with less genetic diversity survived different type of stress better thn nive popultion. The F 1 popultion tht ws selected for fluornthene resistnce showed substntil copper tolernce, wheres seprte group of F 1 fish showed reduced fluornthene resistnce. This study indictes tht reserchers should be cutious in mking ssumptions bout selected popultions. Exposure to n cute stress (e.g., fluornthene) my or my not led to incresed tolernce to tht stress or to other toxicnts in future genertions. Wht is cler is tht when popultion is exposed to n cute stress, the genetic diversity nd genetic composition of tht popultion my chnge. Chnges in popultion s genetic diversity nd composition my either help or hinder tht popultion s surviving similr or different stresses in the future. Only by studying these combined toxicnt (stresses) exposures over multiple genertions will we begin to understnd complex reltionships tht occur in the environment. This study hs lso shown tht P. promels whose prents hd been exposed to n cute fluornthene stress exhibited enhnced tolernce to n cute copper exposure. Whether other tx show the sme response to these two stressors or other stressors remins to be determined. Acknowledgement Funding for this study ws provided in prt by the U.S. Environmentl Protection Agency grnt R to S.I. Guttmn, J.T. Oris, nd D.H. Tylor, Mimi University. REFERENCES 1. Kopp, R.L., S.I. Guttmn nd T.E. 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