genetic ecotoxicology ii: population genetic structure in ...ctheodo/index_files... · 2.5 mm...

Genetic ecotoxicology II: population geneticstructure in mosquito®sh exposed in situ toradionuclides

CHRISTOPHER W. THEODORAKIS 1 and LEE R. SHUGART2�

1Texas A&M University, College Station, TX77483, USA and 2LR Shugart & Associates, Inc., PO Box 5564,

Oak Ridge, TN 37831-5564, USA

Received 1 July 1996; accepted 16 December 1996

In 1977, approximately 250 mosquito®sh (Gambusia af®nis) from a relatively uncontaminated site

(Crystal Springs) were transplanted into a small pond on the Department of Energy Oak Ridge

Reservation which is heavily contaminated with radionuclides (Pond 3513). Starting in 1992, DNA

polymorphism was evaluated using the RAPD (Randomly Ampli®ed Polymorphic DNA) and allozyme

genotype techniques to determine if genetic differentiation had occurred between the two populations.

Fish from a second radionuclide-contaminated population (White Oak Lake) and another unrelated

non-contaminated population (Wolf Creek) were also examined. For the RAPD analyes, 15 RAPD

primers (from a total of 40) were found to produce polymorphic banding patterns in at least two of

the four populations and subsequently were used to produce a total of 142 bands. Data generated by

these RAPD primers indicated an increased genetic diversity in radionuclide-contaminated sites

relative to reference sites. Furthermore, the patterns from six RAPD primers produced a higher

average number of bands when using DNA from radionuclide-contaminated populations than from

non-contaminated, and for three RAPD primers the average number of bands from radionuclide-

contaminated populations was lower. In addition, 17 bands occurred at a higher frequency in the

radionuclide-contaminated compared to the non-contaminated populations. For the allozyme analyses,

it was found that there was a higher precentage of polymorphism and heterozygosity in the

radionuclide-contaminated relative to non-contaminated sites. These ®ndings contribute to our

understanding of the evolutionary effects of contaminant exposure as well as to the development of

population-level biomarkers.

Keywords: population genetics; mosquito®sh; radiation; RAPD; allozymes; evolutionary toxicology.

Introduction

There is inevitably some variation in measurable responses of ®sh or other organisms totoxic stress (Theodorakis et al., 1992). This can be attributed in part to genetic variation.If this is the case, then there is a possibility of natural selection in contaminatedenvironments on genetic variants, which may lead to observable changes in genefrequencies of genetic diversity (Lavie and Nevo, 1982; Nevo et al., 1986; Ben-Schlomoand Nevo, 1988; Strittholt et al., 1988; Gillespie and Guttman, 1989; Kopp et al., 1992;Guttman, 1994; Anderson et al., 1994).

Ecotoxicology 6, 335±354 (1997)

0963±9292 # 1997 Chapman & Hall

�To whom correspondence should be addressed.

It has been suggested that changes in population genetic structure may precedeecological damage that either (a) results from declines in recruitment or survivorship(`ecological effects'; Benton and Guttman, 1992), or (b) results in decreased potentialfor adaptation and increased probabilities of local extinctions (`evolutionary effect';Bickham and Smolen, 1994). These types of effects are of special concern because theycan manifest themselves long after the source of contamination has been eliminated(Anderson et al., 1994). Therefore, monitoring changes in population genetic structurecan be a valuable component of ecological risk assessments by providing a type ofpopulation-level biomarker of contaminant exposure or effects (Suter, 1990).

In this study, the potential for toxic stress to affect population genetic structure wasexamined in four populations of mosquito®sh (Gambusia af®nis) living on or near theDepartment of Energy Oak Ridge Reservation. These populations are particularly suitedfor such studies because in 1977 ®sh from a non-contaminated site (Crystal Springs)were transplanted to a pond heavily polluted with radionuclides (Pond 3513). In aprevious study, Theodorakis et al., (1996) examined the relationship between DNAdamage and reproductive parameters in these same populations and evidence waspresented which suggested that individuals with more DNA damage (strand breakage)had lower reproductive success. If strand breakage is a re¯ection of susceptibility togenotoxicants, then these ®ndings imply that ®sh which are less susceptible may have aselective advantage in contaminated environments. This selection may be manifested bydifferences in population genetic structure between contaminated and non-contaminatedpopulations.

Population genetic structure was examined using RAPD (Randomly Ampli®edPolymorphic DNA) and allozyme techniques. Although the latter technique has beenused extensively for this purpose, the former has received less attention. Nevertheless,the RAPD technique has several advantages over other methods to assess polymorphismin DNA. First, only minute amounts of DNA are needed. Second, an a priori knowledgeof genomic sequences is not required. Third, the technique has the potential to accessmany loci, thus leading to identi®cation of loci that were not previously known to beunder contaminant-selective pressure. Finally, RAPD primers amplify from invertedrepeat sites within the DNA. This is important because certain DNA sequences thatcontain inverted repeats (e.g. transposons) have been shown to be responsive to DNAdamaging agents (Doring and Starlinger, 1986).

Although the RAPD method may be appropriate for genotoxic study, theexperimentalist should be aware of drawbacks. The technique is sensitive to reactionconditions that sometimes result in the production of non-reproducible bands (Williamset al., 1991; Hadrys et al., 1992). If, however, reaction conditions are rigidlystandardized and care is taken to identify non-reproducible bands, the banding patternscan be highly reproducible (Dinesh et al., 1995; personal observation). Also, thespeci®c locations and identities of RAPD-ampli®cation sites within the DNA are notknown a priori, which makes interpretation of the adaptive signi®cance of such markersdif®cult.

The objective of this study was to determine if exposure to radionuclides, a specialclass of genotoxicants, causes changes in population genetic structure (e.g. geneticdiversity and=or genotype frequencies). An ancillary goal was to evaluate theeffectiveness of the RAPD and allozyme techniques to detect such changes. It ishypothesized that genetic diversity will decrease in genotoxicant-contaminated

336 Theodorakis and Shugart

populations. Futhermore, it is predicted that certain genotypes will have a selectiveadvantage in contaminated populations (in terms of fecundity), and that the frequencyof these genotypes will be greater in the contaminated populations.

It should be noted that the long-term goals of this and other studies by the authors(Shugart and Theodorakis, 1994, 1996; Theodorakis et al., 1996) are (a) to developpopulation-level biological markers (and indicators) of contaminant exposure andeffects, and (b) to understand the evolutionary consequences of exposure to environ-mental contamination, especially as they may affect the process of natural selection.

Methods and materials

Chemicals

The following materials were used: Taq polymerase with assay buffer, from PromegaCorp. (Madison, WI); DNA molecular length markers (bacteriophage ÖX174 DNA,Hae III digest) from Promega or Sigma Chemical Corp. (St Louis, MO); agarose, Tris-HCl, EDTA, boric acid, phenol, and ethidium bromide from Gibco-BRL Inc.(Gaithersburg, MD); deoxynucleotides (dATP, dCTP, dGTP, and dTTP) from BoehringerManheim (Indianapolis, IN); bovine serum albumin (BSA) from Idaho Technology(Idaho Falls, ID); chloroform and ethanol from J.T. Baker (Phillipsburg, NJ); RAPDoligonucleotide primers OPD1 through OPD20 from Operon Technologies (Alameda,CA) and primers UBC1 through UBC20 from the Nucleic Acid and Protein ServiceUnit, Biotechnology Laboratory, University of British Columbia (Vancouver, BC). Thesources of reagents for allozyme starch gel electrophoresis are reported in Kramer et al.(1989).

Study sites=specimen collection

The four populations examined in this study were from two radionuclide-contaminatedsites situated on the US Department of Energy reservation in Oak Ridge, TN: Pond 3513and White Oak Lake; and two reference sites located off the reservation: Crystal Springsand Wolf Creek. A detailed description of these sites as well as the method for thecollection of adult, female mosquito®sh, the preservation of samples and isolaton ofDNA from the blood, the determination of brood size and relative fecundity are found inTheodorakis et al. (1996).

RAPD analysis

RAPD reactions were carried out using either a DNA Thermal Cycler 480 (Perkin-ElmerCetus Corp., Norwalk, CT) or an Air Thermo Cycler 1605 (Idaho Technologies, IdahoFalls, ID). For each particular RAPD primer, all populations were examined using thesame apparatus. For the Perkin-Elmer apparatus, the reaction mixture consisted of2.5 mM MgCl2, 100 ìM of each nucleotide triphosphate, 15 ng primer, 10 ng DNA, 1Unit Taq polymerase and a 1=10 volume of the assay buffer supplied by Promega Corp.in glass double-distilled water. Reactions were carried out in a voume of 25 ìl followingthe ampli®cation protocol modi®ed from Williams et al. (1991). For the Idaho apparatus,the reaction mixture consisted of 2.5 mM MgCl2, 10 mg ml

ÿ1 BSA, 100 ìM of eachnucleotide triphosphate, 12 ng primer, 20 ng DNA and 0.6 units Taq polymerase in glassdouble-distilled water. The reactions were carried out in a volume of 10 ìl following the

Population genetics in mosquito®sh 337

ampli®cation protocol supplied by the manufacturer (Skroch and Nienhuis, 1992). All ofthe above procedures were carried out using sterile technique.

Ampli®cation products were loaded onto a 1.5% agarose gel with 0.5 ìg mlÿ1ethidium bromide and subjected to electrophoresis at 2.5 V cmÿ1 for 3 h. An aliquot ofmolecular length standards (bacteriophage ÖX174 DNA digested with Hae III) was runalongside the samples for determination of realtive molecular length (Weir, 1990). Afterelectrophoresis, the gels were photographed under UV light.

RAPD data analysis

The nomenclature for each primer and for each band was as follows. The name of eachprimer consisted of the abbreviation of the manufacturer followed by a number assignedby the manufacturer to identify each primer. Operon Technologies produces several setsof primers. For this experiment, set D was used, so the primers in this set were identi®edas OPD. The Univeristy of British Columbia primers were identi®ed as UBC. Each bandis indicated by the primer which was used to amplify it, followed by its molecular lengthin subscript. For example, band UBC2830 is a fragment 830 nucleotides long produced byprimer UBC2.

The data generated from bands on the gels included: (a) the average number of bandsper individual; (b) the frequency of each band (i.e. the proportion of individuals in apopulation for which each band is produced); and (c) genetic diversity. Number ofbands were scored because, for other types of DNA ®ngerprinting, this parameter hasbeen associated with heterozygosity (Lynch, 1990). Differences between populations interms of average number of bands were tested using the Kruskall-Wallace test withmultiple comparisons; and in terms of band frequencies using a ÷2 test.

The RAPD technique is relatively new and no one standard method is routinely usedfor determining genetic diversity with this procedure. Nevertheless, for comparison, twodifferent but accepted methods were utilized. They were: (a) the similarity index (S),which measures average similarity of banding patterns between all possible pairs ofindividuals in the population (Lynch, 1990); and (b) the nucleon diversity index (h: Nei,1987). Differences between populations were tested by calculation of a standard normaltest statistic using the variance of S and h (Lynch, 1990; Nei, 1987). Because S isinversely proportional to genetic diversity, genetic diversity is reported as thedissimilarity index (D), where D 1 ÿ S (Lynch, 1990). The D and h indices rangebetween 0 (population completely homogeneous) and 1 (populations are completelyheterogeneous).

Selection coef®cients were calculated for ®sh with or without particular bands, wherethe selection coef®cient 1 ÿ w. The relative ®tness of ®sh without these bands (w)was calculated by dividing the average fecundity of ®sh with these bands by theaverage fecundity of ®sh without the bands. The correlations between selectioncoef®cients and band frequency for ®sh from Pond 3513 were calculated and examinedwith Kendall's test.

Genetic distances between populations were calculated using the formula for Roger'sgenetic distance (Nei, 1987) utilizing band frequencies instead of allele frequencies.This index has a value of 0 to 1 when the populations are completely identical andcompletely different, respectively. Testing for differences between pair-wise distancescan be problematic (Nei, 1987), therefore, the distances for each locus were treated as a


sample, and the differences between pair-wise distances were then tested using aStudent's t-test, as suggested by Nei (1987).

Dendrograms were constructed employing the unweighted pair-group method usingan arithmetic average (Weir, 1990).

Allozyme analysis

The methods for allozyme analysis were as described in Diamond et al. (1989). Brie¯y,the whole carcass of each ®sh was homogenized with a glass rod in approximately50±150 ìl of buffer (10 mM Tris, 1 mM EDTA, 50 mM NADP, pH 7.0) and centrifugedat 12 000 rpm in a micro centrifuge for 45 s, loaded into a 12.5% starch gel andsubjected to electrophoresis for at least 10 h. Conditions for electrophoresis and stainingof each enzyme can be found in Harris and Hopkinson (1976), Ayala et al. (1972) andSelander et al. (1971). Allelic variants of each enzyme were scored as fast (F) or slow(S), depending on their relative mobility. Homozygotes were given the connotation FF orSS, while heterozygotes were depicted as FS.

Genetic variation was measured as degree of heterozygosity or polymorphism.Differences in heterozygosity were tested by calculating the variance for the hetero-zygosity estimate (Weir, 1990) and using this variance to calculate a standard normaltest statistic. Differences in polymorphism between populations were tested using a ÷2

test. Genetic distances (D) between populations were calculated usign Roger's distance(Nei, 1987). The differences between distances were tested and dendrograms wereconstructed as above.

Results

Of the 40 different RAPD primers (OPD1 to OPD20 and UBC1 to UBC20) used toscreen for polymorphic banding patterns, only 15 were found to produce banding patternswhich were polymorphic in at least two of the four ®sh populations (Table 1). The resultsusing these 15 RAPD primers are reported here.

Genetic diversity

Genetic diversity within the four ®sh populations, as revealed by the RAPD and allozymeassays, is shown in Fig. 1 and is higher within the radionuclide-contaminated sitesrelative to non-contaminated sites.

RAPD assay. Data in Fig. 1A represent the dissimilarity index (D 1 ÿ S) where S isinversely proportional to genetic diversity. Data from all RAPD primers were pooledtogether to calculate S (Lynch, 1990). When all RAPD primers were pooled, the nucleondiversity index (h) was 1 (indicating each individual ®sh had a unique set of RAPDprimer-speci®c banding patterns). Therefore, the h values were calculated for each RAPDprimer separately and averaged together.

Allozyme assay. The degree of heterozygosity and polymorphism were observed to behigher in the two radionuclide-contaminated populations relative to non-contaminatedpopulations (Fig. 1B).


Genetic distance

The genetic distances were calculated using both the RAPD and allozyme techniques.Data from the RAPD assay and the allozyme NP assay indicated that the tworadionuclide-contaminated populations were more similar to each other than to the non-contaminated populations (Fig. 2A and C). When data from all allozyme loci wereincluded (Fig. 2B), the population in the radionuclide-contaminated Pond 3513 were mostsimilar to the reference population in Crystal Springs.

A

B

Dis

sim

ilarit

y In

dex

(D)

Het

eroz

ygos

ity

Pol

ymor

phis

mN

ucle

on D

iver

sity

Inde

x (h

)

0.4

0.3

0.2

0.1

0

1

0.8

0.6

0.4

0.2

0

0.8

0.4

0.2

0White Oak Lake

Pond 3513 CrystalSprings

WolfCreek

Heterozygosity

Polymorphism

DissimilarityNucleon Diversity

0.04

0.03

0.02

0.01

0

A B C D

A A B C

a b ca

a b c d

Fig. 1. Genetic diversity measurements in mosquito®sh populations from radionuclide-

contamined (Pond 3513 and White Oak Lake) and non-contamined (Crystal Springs and

Wolf Creek) sites using, A: the RAPD assay; or B: the allozyme assay. Bars labelled with

the same letters are not signi®cantly different (P , 0.05).


A Pond 3513

White Oak Lake

Crystal Springs

Wolf Creek

Roger’s Genetic Distance0.30 0.25 0.20 0.15

B Pond 3513

White Oak Lake

Crystal Springs

Wolf Creek

Roger’s Genetic Distance0.40 0.30 0.20 0.10

Pond 3513

White Oak Lake

Crystal Springs

Wolf Creek

Roger’s Genetic Distance

C

0.08 0.07 0.06 0.05 0.04 0.03

Fig. 2. Dendrograms (unweighted pair-group method average) representation of genetic

distance between mosquito®sh populations from radionuclide-contamined (Pond 3513 and

White Oak Lake) and non-contamined (Crystal Springs and Wolf Creek) sites. Data

generated using, A: the RAPD assay; B: the allozyme assay (12 loci); or C: the alloyzyme

assay with nucleoside phosphorylase locus only (C).


Genotype distributions

Three different metrics used to describe genotypes produced by allozyme and RAPD datawere: (a) genotype frequencies, (b) RAPD band frequencies, and (c) number of RAPDbands.

Genotype frequencies. Twelve loci were examined: malate dehydrogenase (MDH, threeloci: MDH1, MDH2, MDH3); fumarase (FUM); isocitrate dehydrogenase (IDH);nucleoside phosphorylase (NP); hydroxybutarate dehydrogenase (HBDH); lactatedehydrogenase (LDH); adenosine deaminase (ADA); phosphoglucomutase (PGM);glucose-6-phosphate dehydrogenase (GPD); and lysyl-alanine peptidase (PEP). Theresults for individual allozyme loci revealed that the levels of heterozygosity werevery low. If a locus was polymorphic, there were only two genotypes present: FSand SS (PEP) or FS and FF (all other loci). The nucleoside phosphorylase locus wasthe exception. The percentage of NP heterozygotes at this locus was an order ofmagnitude higher than any of the other loci, in addition, all three genotypes werepresent. Therefore the NP locus was analysed separately. As demonstrated in Fig. 3, theNP heterozygotes were present at a higher frequency and the SS homozygote waspresent at a lower frequency in the two radionuclide-contaminated sites (P , 0.05, ÷2

test).The only other locus which indicated a difference between the radionuclide-

contaminated and non-contaminated sites was HBDH, with heterozygosities in WhiteOak Lake and Pond 3513 of 0.04 and 0.067, respectively (no HBDH heterozygoteswere observed in the other sites). The other polymorphic loci found in populations fromPond 3513 were FUM and GPD, with heterozygosities of 0.085 and 0.067, respectively.

Fast/Fast Fast/Slow Slow/Slow

Genotype

Freq

uenc

y

1.0

0.8

0.6

0.4

0.2

0.0

A,B A,B A A A BB CA BC D

Fig. 3. Nucleoside phosphorylase genotype frequencies in mosquito®sh populations from

radionuclide-contamined (Pond 3513 and White Oak Lake) and non-contamined (Crystal

Springs and Wolf Creek) sites. `Fast' and `slow' refer to relative electrophoretic mobility of

alleles on starch gels. Bars labelled with the same letters are not signi®cantly different (P ,0.05, ÷2 test). White Oak Lake ; Pond 3513 ; Crystal Springs ; Wolf Creek .


In White Oak Lake, the polymorphic loci included MDH1 (0.082 heterozygotes),MDH2 (0.04), LDH (0.082), and ADA (0.061). In Crystal Springs, NP was the onlypolymorphic locus. In Wolf Creek, the polymorphic loci were MDH1 and LDH, withheterozygosities of 0.076 and 0.051, respectively. Genotypic distributions of all lociwere in agreement with Hardy-Weinberg expectations.

RAPD band frequency. There were a total of 142 bands produced by the 15 RAPDprimers used in this analysis. Seventeen of these bands increased in frequency in both ofthe radionuclide-contaminated populations relative to both of the non-contaminatedpopulations (Table 2). For purposes of discussion, these bands will be referred to as`contaminant-indicative bands'. Four of the bands decreased in frequency in bothcontaminated sites relative to both non-contaminated sites.

Number of RAPD bands. The average number of bands was greater in radionuclide-contaminated populations than for non-contaminated populations for six RAPD primers(Fig. 4A). Again, for purposes of discussion, these RAPD primers will be referred to as`contaminant-indicative primers'. The average number of bands was lower in radio-nuclide-contaminated populations for three of the RAPD primers (Fig. 4B). When thenumber of bands produced by all 15 of the RAPD primers were pooled, there were smallbut signi®cant differences between sites, with radionuclide-contanimated sites havinghigher numbers of bands than non-contaminated sites (Fig. 4C). In addition, when thenumber of RAPD bands present in populations at the various sites was compared withallozyme data (Fig. 5), it was observed that heterozygotes had more RAPD bands thanhomozygotes (P , 0.05, Wicoxon rank-sum test).

Genotype and ®tness

NP heterozygotes had a higher fecundity than homozygotes for both Pond 3513 andWhite Oak Lake, but not for either of the reference sites (P , 0.05, Wilcoxon Rank-Sumtest; Fig. 6). There was also a positive correlation between number of heterozygous lociand fecundity for both of the radionuclide-contaminated sites (Pond 3513: r2 0.395,P , 0.001; White Oak Lake: r2 0.384, P 0.05, Kendall correlation test). In thereference sites no individual had more than one heterozygous locus, so no suchcorrelations could be performed.

In Pond 3513, ®sh which possessed eight of the `contaminant-indicative bands' hadhigher fecundity than ®sh which did not (Fig. 7A). For three other bands, there was ageneral trend in this direction, but the differences were statistically signi®cant only at

Table 1. List of RAPD primersa that produced polymorphic banding patterns in the mosquito®sh

(Gabusia af®nis)

OD RAPD primers

OPD2 OPD7 OPD8 OPD11 OPD12 OPD13 OPD18 OPD20

UBC RAPD primers

UBC1 UBC2 UBC4 UBC6 UBC9 UBC12 UBC16

aSource of RAPD primers: Operon Technology (OD); Univeristy of British Columbia (UBC).Sequences can be obtained from source.


P < 0.10. Similar results were found in White Oak Lake (Fig. 7B). However, for bothradionuclide-contaminated sites, individuals with band UBC41610 had a lower fecunditythan did ®sh without this band, even though this band was elevated in frequency at bothsites. This is opposite of what was observed for other `contaminant-indicative bands'. Inthe Crystal Springs population (Fig. 7C) one band was noted for which fecundity wassigni®cantly greater for ®sh with vs. without a `contaminant-indicative band'. Fishwithout band UBC9920 on the other hand, had a higher fecundity than did ®sh with thisband which is the opposite of what was found at the radionuclide-contaminated sites.Similar results were observed for bands UBC560 and UBC161020 in the Wolf Creekpopulation (Fig. 7D).

The eight `contaminant-indicative bands' that correlated with higher fecundity in the®sh populations of Pond 3515 and White Oak Lake (Fig. 7A and B) were used tocalculate selection coef®cients. A plot of the selection coef®cients versus the frequency

Table 2. Frequency of selected RAPDa bands for two radionuclide-contaminated (Pond 3513 and

White Oak Lake) and two non-contaminated (Crystal Springs and Wolf Creek) mosquito®sh

populationsb

Populationc

RAPD band Pond 3513 White Oak Lake Crystal Springs Wolf Creek

A. Frequency in radionuclide-contaminated . non-contaminatedOPD21586 1.00

1 0.971 0.062 0.573

OPD21060 1.001 1.001 0.092 0.003

OPD71721 0.311 0.181 0.002 0.022

OPD71390 0.621 0.362 0.213 0.203

OPD13428 0.961 0.742 0.593 0.004

OPD20605 0.201 0.672 0.063 0.113

OPD20433 0.781 0.482 0.093 0.004

UBC42055 0.911 0.742 0.523 0.104

UBC41610 0.711 0.771 0.312 0.272

UBC41407 0.471 0.531 0.342 0.133

UBC6575 0.441 0.642 0.213 0.313

UBC6455 0.201 0.231 0.002 0.002

UBC9921 0.771 0.522 0.453 0.034

UBC121269 0.571 0.482 0.003 0.003

UBC121158 0.721 0.192 0.003 0.003

UBC12861 0.131 0.292 0.003 0.023

UBC161016 0.761 0.781 0.362 0.432

B. Frequency in non-contaminated . radionuclide-contaminedOPD8560 0.85

1 0.672 0.933 0.973

OPD13787 0.261 0.211 1.002 0.972

OPD13677 0.481 0.531 0.832 0.973

OPD13263 0.021 0.001 0.832 0.972

aBands which showed consistent differences in band frequency between sites are reported here.bSample sizes for populations were: Pond 3513, 40; White Oak Lake, 40; Crystal Springs, 38; Wolf Creek, 35.cValues with same numerical superscrips are not signi®cantly different (P . 0.05, ÷2 test).


of these bands produced a positive correlation (Fig. 8). This correlation was greater inPond 3513 (P 0.03; Fig. 8A) than in White Oak Lake (P 0.10; Fig. 8B).

For many of the `contaminant-indicative primers' (i.e. RAPD primers for whichthe average number of bands per individual was greater in the two radionuclide-contaminated sites), there was a positive correlation between fecundity and number ofbands (i.e. individuals with more bands had a higher fecundity) at P < 0.05(Table 3). This was true for six RAPD primers in White Oak Lake, four in Pond3513, and two in Crystal Springs. For RAPD primers OPD2 and UBC9, the trendwas the opposite of that for at least one of the radionuclide-contaminated sites. ForRAPD primer OPD13, there was a negative correlation in White Oak Lake but apositive correlation in Crystal Springs. Interestingly, for this RAPD primer, thecontaminated populations showed on average fewer bands per individual than at thereference sites.

A A A

A A B C

14

12

10

8

6

4

2

0

12

10

8

6

4

2

0

10

8

6

4

2

0

100

80

60

40

20

0OPD8 OPD12 OPD13 White Oak Lake

Pond 3513 CrystalSprings

Wolf Creek

OPD2 OPD7 OPD20 UBC4 UBC9 UBC12

Pond 3513White Oak LakeCrystal SpringsWolf Creek

A. Contaminated > Reference A. (cont')

B. Reference > Contaminated C. All Primers Pooled

Ave

rage

num

ber

of B

ands

per

Indi

vidu

al

A B C B C D A B B A A B C A A B C A A B C

A A B BA A

B B A B C C

Fig. 4. Average number of bands produced by nine different RAPD primers for mosquito®sh

populations from radionuclide-contamined (Pond 3513 and White Oak Lake) and non-contamined

(Crystal Springs and Wolf Creek) sites. RAPD primers selected on demonstrated differences between

radionuclide-contaminated and non-contamined populations. Bars and error bars represent medians and

quartiles, and similar letters indicate no signi®cant differences. Data for RAPD primers, A: that

indicate a higher average number of bands in the radionuclide-contaminated sites; B: that indicate a

lower average number of bands in the non-contamined sites; and C: when all RAPD primers were

pooled together.


WhiteOak Lake

Pond 3513 Crystal Springs Wolf Creek

100

80

60

40

20

0

Num

ber

of R

AP

D B

ands

Heterozygote

Homozygote

[15] [32] [16] [31] [11] [29] [4] [36]

Fig. 5. Number of ampli®ed RAPD bands displayed by mosquito®sh homozygous or

heterozygous for at least one locus. Bars and error bars are medians and quartiles. Numbers

above bars are sample size.

Pond 3513 White Oak Lane

HeterozygoteHomozygote

Fec

undi

ty (

Bro

od S

ize/

Bod

y Le

ngth

)

2.5

2

1.5

1

0.5

0

Fig. 6. Fecundity of mosquito®sh from radionuclide-contamined (Pond 3513 and White Oak

Lake) homozygous or heterozygous for the nucleoside phosphorylase (NP) genotypes. Bars

and error bars indicate medians and quartiles. No signi®cant differences were found in non-

contamined populations.


Fec

undi

ty (

Bro

od S

ize/

Bod

y Le

ngth

)

2

1.5

1

0.5

0

2.51.4

1.2

1

0.8

0.6

0.4

0.2

0

**D. Wolf Creek

[12] [28] [17] [23]

UBC6 UBC16560 1020

B. White Oak Lake

********

[19]

[29]

[17]

[31]

[41]

[7]

[36]

[12]

[32]

[16]

[34]

[14]

[37]

[11]

[23]

[25]

[9]

[39]

[14]

[34]

[37]

[11]

OP

D2

1590

OP

D7

1390

OP

D8

560

340

OP

D13

OP

D20 610

910

1610

UB

C2

UB

C4

UB

C12

1270

1160 860

1020

UB

C12

UB

C12

UB

C16

OP

D20 430

1610

UB

C4

UB

C4

440

580

460

UB

C6

UB

C6

UB

C9

920

1270

1160

1020

UB

C12

UB

C12

UB

C16

OP

D20

OP

D7

610

1150 OPD7

1150OPD13 510

UBC4 1210

UBC4 690

UBC9 1290

C. Crystal Springs[16][19]

[7][28]

[29][6] [29][6] [29][6]

**

A. Pond 3513 With Band Without Band

* * * * * ***

[26]

[14]

[8]

[32]

[31]

[9]

[34]

[14]

[39]

[9]

[17]

[23]

[11]

[37]

[30]

[9]

[22]

[18]

[28]

[12]

[30]

[10]

3

2.5

2

1.5

1

0.5

0

2

1.5

1

0.5

0

Fig. 7. Fecundity of mosquito®sh populations from radionuclide-contamined (Pond 3513 and White Oak Lake) and non-contamined (Crystal Springs

and Wolf Creek) sites with and without contaminated±indicative RAPD bands. � indicates P < 0.05 (Ho: No difference between ®sh with and withoutband; Wilcoxon Rank-sum test).

Popula

tion

gen

eticsin

mosq

uito

®sh

347

Discussion

Population genetic structure data derived from the RAPD and allozyme assays revealdifferences between radionuclide-contaminated and non-contaminated populations andsuggest that changes have occurred since Pond 3513 was colonized from Crystal Springsin 1977. In addition, the genetic similarity observed between Pond 3513 and White OakLake suggest that these differences may be due to radionuclide-induced selection.

It should be noted that the differences between radionuclide-contaminated and non-contaminated populations may not be due solely or even primarily to radionuclidecontamination. This is a preliminary investigation and the number of populationsstudied was small, therefore, other processes may very well be involved in producingthe changes observed between the various populations. For example, differencesbetween band frequencies could be due to random drift, neutral mutation or selectiondue to some other environmental variable not directly related to radiation exposure.Differences in genetic diversity can also be driven by neutral processes. For instance,relationships have been shown between genetic polymorphism and habitat heterogeneity

0.7

0.6

0.5

0.4

0.3

0.2

0.1

00 0.2 0.4 0.6 0.8 1

Band Frequency

0 0.2 0.4 0.6 0.8 1

0.2

0.15

0.1

0.05

0

A. Pond 3513

r2 5 0.423P 5 0.423

B. White Oak Lake

r2 5 0.333P 5 0.10

Sel

ectio

n co

effic

ient

..

.

..

.

.

..

.

.

..

.

.

.

.

.

..

.

Fig. 8. Correlation between frequency of `contaminated-indicative' RAPD bands vs.

selection coef®cient for RAPD bands. No signi®cant correlations noted for either non-

contamined site.


(DeMeeus et al., 1993) and between genetic variability and population density (Nei etal., 1975). Alternatively, the mosquito®sh population in Pond 3513 could haveundergone a bottleneck upon introduction from Crystal Springs. It has been argued inthe past that bottlenecks should decrease variability (Nei et al., 1975), but some recentevidence suggests the opposite (Wills and Orr, 1993), both for single-locus (Leberg,1992) and quantitative traits (Lopez-Fanjul and Villaverde, 1989).

Several lines of evidence which would argue against these neutral hypotheses are:

(i) Measures of genetic diversity (Figs 1 and 2) indicate that the contaminatedsites are more similar to each other than to the non-contaminated sites, eventhough the Crystal Springs (reference) site is ancestral to Pond 3513(radionuclide-contaminated).

(ii) Relationships observed between genotype and ®tness in the contaminated sitesare not re¯ected in the non-contaminated sites. These include differences infecundity between ®sh with and without contaminant-indicative bands (Fig. 7),correlations between number of bands and fecundity for `contaminant-indicativeprimers' (Table 3), and correlations between band frequency and selectioncoef®cients (Fig. 8).

(iii) Recently published studies demonstrated a relationship between genotype andDNA strand breakage (an effect of radiation exposure) which parallels therelationship between fecundity and genotype (Theodorakis et al., 1996).

(iv) Other studies utilizing the RAPD assay that indicate an increase in geneticdiversity in organisms other than mosquito®sh at contaminated sites on the OakRidge Reservation (K.L. Lee, Oak Ridge National Laboratory, unpublisheddata; Nadig, 1996).

Table 3. Kendall correlation coef®cients r and probability values (P) between number ofRAPD bands and fecundity for mosquito®sh from two radionuclide-contaminated populations

(White Oak Lake and Pond 3513) and two non-contaminated (Crystal Springs and Wolf

Creek) populations

Population

White Oak Lake Pond 3513 Crystal Springs Wolf Creek

Primer r Pa r P r P r P

OPD2 0.35 0.03 nsb ns ÿ0.44 ,0.01 ns nsOPD7 ns ns 0.34 0.03 ns ns ns ns

OPD8 0.40 0.03 0.21 0.08 ns ns ns ns

OPD13 ÿ0.50 0.01 ns ns 0.35 0.05 ns nsOPD20 0.53 ,0.01 ns ns ns ns ns nsUBC2 0.48 ,0.01 ns ns ns ns ns nsUBC4 0.32 0.07 ns ns 0.35 0.05 ns ns

UBC6 ns ns 0.32 0.01 ns ns ns ns

UBC9 ns ns 0.35 0.01 ns ns ÿ0.76 0.02UBC12 0.50 ,0.01 0.39 ,0.01 0.64 0.03 ns ns

aProbability (á) level for the test of Ho: r 0.bNot signi®cant.


(v) Current studies indicate that when mosquito®sh from a non-contaminated sourceare caged in Pond 3513, those which posses `contaminant-indicative bands' have agreater chance of survival than those that do not (unpublished data).

Taken together, these observations imply that radionuclide exposure has an effect onpopulation genetic structure and genetic diversity.

The ®nding of a higher genetic diversity in the radionuclide-contaminated populationsis the reverse of what was hypothesized initially (see Introduction) and what has beenreported in other studies (Nevo et al., 1986; Strittholt et al., 1988; Kopp et al., 1992).At present, de®nitive explanations for the occurrence of higher diversity are unknown,but possibilities include: (a) genomic rearrangements, which are known to be inducedby radiation exposure (Doring and Starlinger, 1986) or other stressors (Cullis, 1990);and (b) differences in life history (Scribner et al., 1992), reproductive (Chesser et al.,1984) or population dynamic=demographic processes (Feder et al., 1984). Past studies(Lavie and Nevo, 1982; Nevo et al., 1986; Ben-Schlomo and Nevo, 1988; Strittholt etal., 1988; Gillespie and Guttman, 1989; Kopp et al., 1992) have focused on structuralgenes (i.e. allozymes), while many RAPD ampli®cation sites may be located in non-coding regions. Thus discrepancies between the present study and other studies may beexplained by differential gene structure and function.

However in the present study, allozyme heterozygosity was also higher in theradionuclide-contaminated populations, again in contradiction to previous studies. Acontributing factor to this apparent discrepancy may be the very low levels ofheterozygosity of the Oak Ridge mosquito®sh populations compared to populationsexamined in these other studies. An exception was the NP locus, for which thepercentage heterozygosity was an order of magnitude greater than any other loci. Forthis locus, there was higher percentage heterozygosity in the radionuclide-contaminatedsites (Fig. 3), and these heterozygotes had higher fecundity than the homozygotes(Fig. 6). The nucleoside phosphorylase enzyme is involved in nucleotide synthesis, animportant biochemical pathway associated with DNA repair, and nucleotide synthesisrates increase in response to gentoxicant exposure (Kunz and Kohalm, 1991).Regardless of the reasons for increased genetic diversity in contaminated habitats, thisparameter may provide a population level biomarker of contaminant exposure.

Other potential population-level biomarkers that re¯ect a difference betweenreference and contaminated populations include average number of bands per individualand frequency of certain bands. In addition, there may be some relationship betweennumber of RAPD bands and heterozygosity, because ®sh with at least one heterozygouslocus had more bands than completely homozygous ®sh (Fig. 5). Furthermore, for manybands and RAPD primers, there were relationships between ®tness and genotype thatwere not re¯ected in the reference populations. The nature of the relationship betweengenotype and fecundity may vary between bands and RAPD primers (Table 4).Interestingly, in some cases the relationships between fecundity and genotype inreference sites were the opposite of those in radionuclide-contaminated sites (see bandsUBC6560, UBC91290, and UBC161020, Fig. 7; and RAPD primers OPD2, OPD13 andUCB9, Table 2). The speci®c relationship for each RAPD primer and band may dependupon the structure and function of RAPD ampli®cation sites within the DNA, as well asmolecular- and population-level determinants of band presence or absence.

Overall, the frequency of such bands was correlated to the selective coef®cient in


Pond 3513, and to a lesser extent in White Oak Lake, but in neither reference site(Fig. 8). Furthermore, when all RAPD primers were pooled it was found that theaverage number of bands was greater in the radionuclide-contaminated populations(Fig. 4D), and in these populations there was a positive correlation between number ofbands and fecundity (Table 3). These ®ndings strongly support the hypothesis ofcontaminant-induced selection as a possible explanation for differences in bandfrequencies or number of bands.

Another potential indicator of such selection is the genetic distance betweenradionuclide-contaminated and non-contaminated populations (Fig. 2). If no contami-nant-induced selection were occurring, one would expect that the smallest distancewould be between Pond 3513 and Crystal Springs, but RAPD data indicated thesmallest distance was between Pond 3513 and White Oak Lake (Figs. 2A and 2C).Deviations from expected genetic distance relationships at contaminated sites have alsobeen found in other studies (Guttman, 1994), and may be a re¯ection of population-level responses to contamination.

Finally, another notable observation is that both the genetic distribution and thegenotype=®tness relationships for Crystal Springs is intermediate between theradionuclide-contaminated sites and Wolf Creek. Although the relative levels ofcontamination in the two reference sites (Crystal Springs and Wolf Creek) are unknown,

Table 4. Summary of the relationship between genotype and fecundity for various RAPD primers and

bands

Effect of genotype on ®tness in radionuclide-

contaminated populationsb

Genotypic distribution Selective advantage

Selective

disadvantage

A. RAPD bandsa

Band frequency greater in radionuclide-

contaminated populations

OPD1590, OPD71390, OPD71160,

OPD20610, UPD20430, VBC2910,

UBC4440, UBC6580, UBC6460,

UBC9920, UBC121270, UBC121160,

UBC12860, UBC161020

UBC41610

Band frequency less in radionuclide-

contaminated populations

OPD8560 none

B. RAPD Primersa

Average number of bands greater in

radionuclide-contaminated populations

OPD2, OPD7, OPD20, UBC2,

UBC4, UBC6, UBC9, UBC12

none

Average number of bands less in

radionuclide-contaminated populations

OPD8 OPD13

aOnly those bands and RAPD primers that displayed a difference between radionuclide-contaminated and non-contaminated populations are presented.bSelective advantage: Higher fecundity for ®sh possessing a particular band or displaying more bands for aparticular RAPD primer, in the radionuclide-contamined populations, but not in either non-contamined population.Selective disadvantage: Lower fecundity for ®sh possessing a particular band or displaying more bands for aparticular RAPD primer, in the radionuclide-contamined populations, but not in either non-contamined population.


it should be noted that the amount of DNA strand breakage in the Crystal Springspopulation was also intermediate beteen the radionuclide-contaminated sites and WolfCreek (Theodorakis et al., 1996). In this regard, radon at a concentration of 100 pCi perlitre has been reported in water obtained from Crystal Springs (J. Larson, Oak RidgeNational Laboratory, unpublished observation).

Conclusion

The results presented in this study provide evidence that radionuclide contamination mayin¯uence evolution of exposed populations through contaminant-induced selection.Furthermore, genetic markers produced by the RAPD or allozyme assays can be useful inidentifying evolutionary effects of contaminant exposure, and may lead to developmentof population-level biomarkers of contaminant exposure or effects. Speci®cally, the datareported here suggest that contaminant-induced selection may have occurred after the ®shfrom Crystal Springs were introduced into Pond 3513. Such `evolutionary effects' are ofconcern because they have the potential to negatively affect population dynamics andlocal extinction rates (Bickham and Smolen, 1994). Also, adaptation to speci®c pollutantsmay be disadvantageous, because individuals more adapted to one class of pollutant maybe more susceptible to other pollutants (Depledge, 1994) or environmental stressors(Trabalka and Allen, 1977). In addition, if population or physiological responses at acontaminated site change over time, it may be possible by applying the approaches andprinciples of evolutionary toxicology to determine the cause (i.e. changes in contaminantconcentrations vs. selection) for resistant individuals. Finally, differential susceptibility togenotoxins is present in the human population (Rudiger, 1991), so investigations into themechanisms of differential genotoxic susceptibility in animals can contribute to humanhealth risk assessment.

Use of RAPD methodology as described here for biomonitoring must be viewedcautiously at this point, because the genetic location or the possible functions of theampli®cation sites from which the RAPD bands are produced is not known withcertainty. Furthermore, effects of random drift as a contributing factor have not beenruled out. The relationship between genotype and fecundity and the fact that bothradionuclide-contaminated sites show similar trends would argue against this hypothesis,but until more studies are done, genetic drift remains a viable alternative hypothesis.

The results presented in this study, although compelling, are only preliminary andmore work needs to be done before the RAPD technique can con®dently be used inroutine environmental monitoring work.

Acknowledgements

I would sincerely like to thank B.G. Blaylock, K.L. Lee and R. Clark for their assistancewith ®eld collections and G.G. McCraken, J. Tuskan, and B.P. Bradley for their critiquesof this manuscript. This project was sponsored in part by an appointment of CWT to theUS Department of Energy Laboratory Cooperative Postgraduate Research TrainingProgram administered by the Oak Ridge Associated Universities and by the US ArmyBiomedical Research and Development Laboratory (DOE project No. 1061-BO47-A1).The Oak Ridge National Laboratory is managed by Lockheed Martin Energy ResearchCorp. for the US Department of Energy under contract DE-AC05-96OR22464. Portions


of this study were presented at the 14th and 15th Annual Meetings of The Society ofEnvironmental Toxicology and Chemistry.

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IntroductionMethods and materialsChemicalsStudy sites/specimen collectionRAPD analysisRAPD data analysisAllozyme analysisResultsGenetic diversityGenetic distanceGenotype distributionsGenotype and fitnessDiscussionConclusionAcknowledgementsReferences

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