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Allozyme genotypes and tolerance to copper stress in Hediste diversicolor (Polychaeta: Nereididae)
Marine Pollution Bulletin 49 (2004) 978–985 www.elsevier.com/locate/marpolbul
Allozyme genotypes and tolerance to copper stress in Hediste diversicolor (Polychaeta: Nereididae) Massimiliano Virgilio *, Marco Abbiati Centro Interdipartimentale di Ricerca per le Scienze Ambientali in Ravenna and Dipartimento di Biologia Evoluzionistica e Sperimentale –– University of Bologna, Via S. Alberto 163, I-48100, Ravenna, Italy
Abstract This study analysed the occurrence of genotypic shifts in laboratory populations of Hediste diversicolor (Polychaeta: Nereididae) exposed to copper stress. Specimens of H. diversicolor were collected at three sites, up to 10 km apart, in the estuarine area of the Pialassa lagoons (North Adriatic Sea, Italy) and were used in acute toxicity tests. Specimens were assigned to copper exposure (0.34 mg/l Cu2+) or control conditions. Each combination of Treatment and Site was replicated in two tanks containing 35 specimens of H. diversicolor. The genotypic structure of both dead and survived specimens was analysed by allozyme electrophoresis at six loci (ALD, FH, HBDH LDH, PGI, SDH). Under copper exposure, specimens with the genotypes ALD100/100 and PGI102/102 had significantly lower mortalities than other genotypes Results were consistent across the three sites, suggesting that, under laboratory conditions, effects of copper stress on H. diversicolor is related to individual genotypes at ALD and PGI loci. 2004 Elsevier Ltd. All rights reserved. Keywords: Hediste diversicolor; Toxicity tests; Copper; Tolerance; Selection; Allozymes
1. Introduction Responses of organisms to contaminants can be a continuum of biochemical, physiological or genetic changes, both at population and at community levels (Troncoso et al., 2000). Survival and reproductive capabilities of individuals were shown to be widely affected by their tolerance to contaminants (Grant et al., 1989). Variability in tolerance levels observed in populations exposed to several categories of contaminants has been suggested to have genetic bases (Bryan and Hummerstone, 1971; Gamenick et al., 1998). Contaminants were hypothesised to cause adaptive changes in the genetic structure of populations by means of differential survivorship and reproduction of individuals (Gillespie and *
Corresponding author. Tel.: +39 0544 600300; fax: +39 0544 600411. E-mail address: [email protected] (M. Virgilio). 0025-326X/$ - see front matter 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.marpolbul.2004.06.027
Guttman, 1993). However, due to the complex interactions of evolutionary and physiological processes, it is generally difficult to show a correlation between concentrations of contaminants and adaptive responses of impacted populations (Belfiore and Anderson, 2001). Potentially, other factors, unrelated to tolerance, can alter the genetic structure of impacted populations, e.g. founder effects resulting from severe reductions in size of populations or selection on traits unrelated to tolerance (Bickham et al., 2000). Moreover, tolerance may arise through many other alternative mechanisms, including epistatic interactions (Danzmann et al., 1999) and physiologic acclimation (Kovatch et al., 2000). For these reasons tolerance is not always synonymous of genetic adaptation and is not always inherited through a specific allele or set of alleles (Belfiore and Anderson, 2001). Copper contamination has been supposed to represent a major selective force promoting the occurrence
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of tolerant strains in different species of invertebrates (Lavie and Nevo, 1982) and fishes (Chagnon and Guttman, 1989). Copper, an essential element in metabolic processes, enters aquatic environments from many sources, including wastewater of industrial and agricultural activities, and becomes toxic when water concentrations exceed 0.010 mg/l (Shlueter et al., 1997). Hediste diversicolor (O.F. Mu¨ller) successfully colonises heavily polluted estuarine sediments with copper concentrations up to 4000 lg/g dw (Bryan and Hummerstone, 1971). This species absorbs copper from ingested sediments as well as from solution (Bryan and Hummerstone, 1971) and the occurrence of regulatory and/or excretory capabilities has been hypothesised (Ozoh, 1994). Absence of a true pelagic phase in the life cycle of H. diversicolor has been supposed to have a major role in favouring adaptation to stress, promoting strong selection of juveniles in environments contaminated by heavy metals (Bryan et al., 1987). A steady decline in tolerance to copper was described at increasing distances from heavily polluted areas (Grant et al., 1989; Bryan and Hummerstone, 1971). Tolerance was shown to be neither readily gained nor readily lost under laboratory conditions and the occurrence of adaptation processes, through the development of metal tolerant strains, has been hypothesised (Bryan and Hummerstone, 1971). Laboratory toxicity tests showed that resistance to copper stress in H. diversicolor had a heritable component, providing evidence for the genetic bases of tolerance (Grant et al., 1989). Moreover, analysis of H. diversicolor along a contamination gradient (Virgilio et al., 2003), revealed non-random patterns of variation at six allozyme loci. Patterns of polymorphism appeared to be possibly related to levels of contamination by heavy metals, with major differences occurring at the most impacted sites (Virgilio et al., 2003). In this study we tested hypotheses about relationships between exposure to copper stress and genetic responses in H. diversicolor. The main objectives were (a) to assess if, under laboratory conditions, copper tolerance may be related to differences in genotypic patterns of H. diversicolor and (b) to verify the occurrence of consistent genetic responses among different sites.
2. Materials and methods Genotype-tolerance responses were analysed in populations of H. diversicolor colonising the estuarine area of the Pialassa lagoons (44.4673–44.5293 N, 012.2375– 012.2705 E, European Datum 1950). This area, located along the northern Adriatic coast of Italy, is characterised by the presence of two interconnected lagoons and a river estuary (Fig. 1). A detailed description of the characteristics of the area can be found in Virgilio et al. (2003) and references therein. Average concentra-
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Fig. 1. Estuarine area of the Pialassa lagoons (North Adriatic Sea, Italy). Specimens of Hediste diversicolor were collected at Sites 1, 2 and 3.
tions of copper in sediments of the Pialassa lagoons are two to three times higher than background levels (Miserocchi et al., 1990), with concentrations ranging from 11 to 280 lg/g dw (Anconelli et al., 1980). Replicate ABS tanks (400 · 300 · 80 mm), containing 3 l of 10 psu artificial seawater (Red Sea Salt, Red Sea Fish pHarm, Israel), were used for toxicity tests. Tanks were continuously aerated and maintained at a temperature of 20 C. Worms were acclimatized for 72 h before exposures, no mortality was observed during the acclimation periods. Specimens were not fed during the experiments. Copper was added as CuSO4. Preliminary range finding experiments were conducted in order to determine the target concentrations of copper (96 h, 50% mortality). Based on the results of the range finding experiments, a target concentration of 0.34 mg/l Cu2+ was used to produce a relevant mortality of specimens of H. diversicolor in a 96-h acute toxicity test. Specimens of H. diversicolor, collected in May 2002, were assigned to copper exposure (0.34 mg/l Cu2+) or control conditions. In order to assess the generality of genotype-tolerance responses in populations of H. diversicolor from the estuarine area of the Pialassa lagoons, specimens were sampled at three different sites up to 10 km apart (Fig. 1). At each site, about 140 specimens of H. diversicolor were collected. Two tanks for each combination of treatment and site were used as independent replicates for statistical analyses. To allow an estimation of the genotypic variability within each replicate, 35 specimens of H. diversicolor were randomly assigned to each tank. Dead worms were removed at least every 12 h, rinsed with distilled water and then stored in Eppendorf tubes at 80 C for the genetic analyses. At the end of the 96-h
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period, surviving H. diversicolor specimens were sacrificed and processed using the same procedure. Differences in percentages of mortality were tested by means of a two-way ANOVA with Treatment (0.34 mg/l Cu2+ exposure, control) as fixed factor and Site (1, 2 and 3) as random, orthogonal factor (n = 2). Six allozyme loci showing non-random patterns of variation along a pollution gradient (Virgilio et al., 2003) were analysed by means of cellulose acetate electrophoresis: ALD (aldolase, E.C. 4.1.2.13); FH (fumarate hydratase, E.C. 4.2.1.2); HBDH (3-hydroxybutyrate dehydrogenase, E.C. 1.1.1.30); LDH (lactate dehydrogenase, E.C. 1.1.1.27); PGI (glucose-6-phosphate isomerase, E.C. 5.3.1.9) and SDH (sorbitol dehydrogenase, E.C. 1.1.1.14). For each specimen of H. diversicolor, about 100 mg of tissue from the central setigers was homogenised with 100 ll of extracting buffer (Tris–HCl 0.05 M pH 8, TritonX100 1:1000), subjected to electrophoresis and stained following the procedures reported in Pasteur et al. (1988) with slight modifications. Tris– EDTA–Maleic acid (pH 7.8) electrode buffer (Schneppenheim and Mac Donald, 1984) was used in 1:1 and 1:2 dilutions. For each locus the most common allele in the reference population (Site 1) was designated as allele 100. Slower and faster bands on the zymograms, representing other alleles, were given lower and higher numbers corresponding to their relative mobility. Linkage disequilibrium among genotypes at pairs of loci in each site was calculated using a log-likelihood ratio G-statistic, as implemented by the computer package FSTAT 1.2 (Goudet, 1995). Multiple tests were adjusted by the sequential Bonferroni correction (Hochberg, 1988). For each of the replicate tanks exposed to copper (0.34 mg/l Cu2+), observed heterozygosity (e.g. the observed proportion of heterozygotes at each locus) was calculated for both dead and survived specimens after 96 h. Differences in observed heterozygosity were tested by means of a two-way ANOVA with Endpoint (dead, survived) as fixed factor and Site (1, 2 and 3) as random, orthogonal factor (n = 2). For each of the replicate
tanks, the mortality ratios associated to the observed genotypes (number of dead/number of survived specimens with the same genotype) were calculated after 96 h. At each locus, differences in mortality ratios were tested by means of a three-way ANOVA with Site (1, 2 and 3) as random factor, Treatment (0.34 mg/l Cu2+, control) and Genotype as fixed, orthogonal factors (n = 2). Assumption of homogeneity of variances for percentages of mortality, heterozygosity values and mortality ratios was tested by means of CochranÕs C-test. In order to identify possible trends in percentages of mortality, heterozygosity levels and mortality ratios, the Student–Newman–Keuls (SNK) test was used for a posteriori multiple comparison of means.
3. Results Average percentages of mortality over the three sites were 3.0% (S.E. = 1.1) in controls and 41.6% (S.E. = 4.2) in treatments exposed to 0.34 mg/l Cu2+. ANOVA and SNK tests (Table 1) did not reveal differences in percentages of mortality among controls while, in copper exposures, specimens of H. diversicolor from Site 2 showed a significantly higher percentage of mortality (m% = 54.4, S.E. = 1.5) compared to Site 1 (m% = 33.8, S.E. = 0.5) and Site 3 (m% = 36.7, S.E. = 3.3). No linkage disequilibrium was observed out of 58 pairwise comparisons of loci. Average observed heterozygosity values (Hobs) were: 0.149 (S.E. = 0.029) at ALD; 0.073 (S.E. = 0.026) at FH; 0.003 (S.E. = 0.003) at HBDH; 0.100 (S.E. = 0.012) at LDH; 0.303 (S.E. = 0.055) at PGI and 0.084 (S.E. = 0.030) at SDH. In specimens exposed to copper, ANOVA on observed heterozygosity at polymorphic loci ALD, FH, PGI and SDH (Fig. 2) did not reveal significant differences, either between dead and surviving specimens or between sites. Yet, ANOVA showed a significant Site · Endpoint interaction at LDH locus, although SNK did not suggest any alternative to the null hypothesis (Table 2). ANOVA on
Table 1 ANOVA and SNK test for the effects of Treatment (0.34 mg/l Cu2+ exposure, control) and Site (1, 2 and 3) on percentages of mortality of Hediste diversicolor Source of variation
df
MS
F
P
Treatment = T Site = S T·S Residual CochranÕs C-test (C = 0.401 p > 0.05) Transformation (None)
1 2 2 6
4466.02 152.75 100.78 9.33
44.31 16.37 10.80
* ** *
SNK test: Treatment · Site 0.34 mg/l Cu2+: Control: *p
< 0.05;
**p
< 0.01.
Site 1 = Site 3 < Site 2 Site 3 = Site 1 = Site 2
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< 0.05; n.s., not significant. *p
Dead: Site 1 = Site 2 = Site 3 Survived: Site 1 = Site 2 = Site 3
SNK test: Site · Endpoint LDH
2 1 2 6 Site = S Endpoint = E S·E Residual CochranÕs C-test Transformation
0.023 2.49 0.014 3.57 0.004 0.42 0.009 C = 0.559 p > 0.05 None
n.s. n.s. n.s.
0.001 0.52 n.s. 0.000 0.03 n.s. 0.003 1.48 n.s. 0.002 C = 0.639 p > 0.05 None
0.011 2.11 n.s. 0.014 0.49 n.s. 0.028 5.61 * 0.005 C = 0.313 p > 0.05 None
0.006 0.62 n.s. 0.005 2.54 n.s. 0.002 0.22 n.s. 0.009 C = 0.423 p > 0.05 None
0.032 1.93 n.s. 0.001 1.00 n.s. 0.001 0.07 n.s. 0.016 C = 0.679 p > 0.05 Square root
Residual S·E Residual
Denominator for F P F MS
SDH
P F MS
PGI
P F MS
LDH
P MS
F FH
F
P MS
It is often difficult to generalise about the toxicity of heavy metals in aquatic organisms because of the active role that organisms play in regulating bioaccumulation/ detoxification processes and of the influence of physical/ chemical variables such as salinity and temperature (Ozoh, 1994). Several processes were suggested to cause differences in tolerance to copper stress among populations of Hediste diversicolor. Variations in permeability of the body surface could affect accumulation and detoxification processes (Bryan and Hummerstone, 1971; Bryan et al., 1987). Copper accumulation in
ALD
4. Discussion
df
mortality ratios (Table 3) showed a significant Treatment · Genotype interaction at ALD and PGI loci. SNK tests revealed that, under copper stress, mortality ratios associated to genotype ALD100/100 were significantly lower than those associated to genotypes ALD100/102 and ALD102/102, while no differences were observed under control conditions (Fig. 3). Similarly, under copper stress, mortality ratios associated to genotype PGI102/102 were significantly lower than those associated to genotypes PGI100/102 and PGI102/102, while no differences were observed under control conditions (Fig. 4). At PGI locus, there was also a significant Site · Treatment interaction, due to the higher mortality ratios in Site 2. Genetic variability at loci HBDH, FH and SDH was very low, therefore ANOVA on effects of copper stress on heterozygosity and on mortality ratios was not performed.
Source of variation
Fig. 2. Hediste diversicolor. Observed heterozygosity (Hobs) of dead and survived specimens after 96-h exposure to 0.34 mg/l Cu2+. Results are shown at ALD, FH, LDH, PGI and SDH loci. Sites as in Fig. 1.
Table 2 Hediste diversicolor. ANOVA and SNK test for the effects of Site (1, 2 and 3) and Endpoint (dead, survived) on observed heterozygosity values at loci ALD, FH, LDH, PGI and SDH in specimens exposed to 0.34 mg/l Cu2+
M. Virgilio, M. Abbiati / Marine Pollution Bulletin 49 (2004) 978–985
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Source of variation
PGI
ALD
df Genotype = G Site = S Treatment = T G·S G·T S·T G·S·T Residual CochranÕs C-test Transformation
MS
2 0.211 2 0.297 1 2.351 4 0.013 2 0.157 2 0.260 4 0.011 18 0.069 C = 0.4037 p > 0.05 None
P
df
15.97 4.31 9.04 0.19 14.58 3.78 0.16
* * n.s. n.s. * * n.s.
2 0.967 2 2.033 1 8.920 4 0.169 2 0.829 2 1.159 4 0.019 18 0.782 C = 0.435 p > 0.05 None
SNK test: Genotype · Treatment
PGI 2+
0.34 mg/l Cu : Control:
100/102 = 100/100 > 102/102 100/102 = 100/100 = 102/102
PGI
SNK test: Site · Treatment 2+
0.34 mg/l Cu : Control:
LDH
F
MS
**p
P
df
MS
5.74 2.6 7.69 0.22 44.35 1.48 0.02
n.s. n.s. n.s. n.s. ** n.s. n.s.
1 2 1 2 1 2 2 12 C = 0.597 None
0.019 0.01 0.713 0.64 0.109 1.57 1.923 1.72 0.032 0.05 0.070 0.06 0.646 0.58 1.117 0.01 < p < 0.05
SNK test: Genotype · Treatment
ALD 2+
0.34 mg/l Cu : Control:
Site 2 > Site 3 = Site 1 Site 2 = Site 1 = Site 3
Results are shown for PGI, ALD, and LDH loci, *p < 0.05;
F
< 0.01; n.s. = not significant.
102/102 = 100/102 > 100/100 102/102 = 100/100 = 100/102
F
P
Denominator for F
n.s. n.s. n.s. n.s. n.s. n.s. n.s.
G·S Residual S·T Residual G·S·T Residual Residual
M. Virgilio, M. Abbiati / Marine Pollution Bulletin 49 (2004) 978–985
Table 3 Hediste diversicolor. ANOVA and SNK test for the effects of Genotype, Site and Treatment on mortality ratios (number of dead/number of survived specimens with the same genotype)
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0.34 mg/l Cu
4
2+
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ALD
PGI control
0.34 mg/l Cu2+
control
Site 1
Site 1
2 2 0
1 0
4
1 0 2 1 0
4
2 Mean
0
2
1
genotype
100/100
100/102
102/102
100/100
100/102
102/102
100/100
100/102
102/102
100/100
0 100/102
0
102/102
Mean
2
Site 2
mortality ratio
4
Site 3
Site 2
0
Site 3
mortality ratio
2 2
genotype
Fig. 3. Hediste diversicolor. Mortality ratios (number of dead/number of survived specimens with the same genotype) associated to locus ALD. Sites as in Fig. 1.
Fig. 4. Hediste diversicolor. Mortality ratios (number of dead/number of survived specimens with the same genotype) associated to locus PGI. Sites as in Fig. 1.
H. diversicolor has been shown to occur more readily at low salinities (Ozoh, 1990), when the uptake of divalent ions may be favoured both by lower competition from calcium and magnesium ions and potential differences across the body surface (Bryan et al., 1987). Higher temperatures have also been shown to increase the accumulation of copper and were supposed to influence changes of heavy metals tolerances (Ozoh, 1994). In this study, different percentages of mortality were observed among sites after exposure to 0.34 mg/l Cu2+, suggesting the occurrence of differently tolerant strains of H. diversicolor. Differences in local environmental conditions at study sites (salinity variations, temperature ranges, levels of contamination, etc.) may have enhanced the selection/acclimation of physiologically different strains of H. diversicolor. Heterozygous individuals at allozyme loci may have lower metabolic requirements and higher efficiency in several metabolic processes (Koehn, 1989). Different studies showed relationships between single- or multilocus heterozygosity and higher tolerances to heavy metal stressors (Diamond et al., 1991; Troncoso et al., 2000). Authors have suggested that heterozygotes, producing different forms of the same metabolic enzyme, may be better buffered against environmental fluctua-
tions (Diamond et al., 1991). Under the experimental conditions used in this study, tolerance to copper stress in H. diversicolor was not related to levels of heterozygosity. Conversely, exposure to copper resulted in higher survivorship of specimens of H. diversicolor owing specific allozyme genotypes. After copper exposure, mortality ratios associated to ALD100/100 and PGI102/102 genotypes were sensibly lower, supporting the model that higher tolerance to copper stress is related to the occurrence of these genotypes. Results are in agreement with other studies showing that in species such as Hyalella azteca (Duan et al., 2001), Pimephales promelas (Shlueter et al., 1997, 2000), Gambusia affinis (Newman et al., 1989) and G. holbrooki (Diamond et al., 1991), tolerance was not associated to heterozygosity levels, but to specific genotypes. Glucose-6-phosphate-isomerase (PGI) has been widely studied, both for its role in the metabolic processes and for its high levels of polymorphism (Troncoso et al., 2000). Previous studies on genetic adaptation to contaminants showed that PGI genotypes are frequently associated to tolerance responses to specific stressors, such as PAH (Shlueter et al., 2000), cadmium (Lavie and Nevo, 1986), mercury (Mulvey et al., 1995), zinc (Lavie and Nevo, 1982) or general levels of pollution (Patarnello et al., 1991).
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Copper exposure resulted in differential survivorship of individuals with different PGI genotypes in species such as Monodonta turbinata, M. turbiformis (Lavie and Nevo, 1982), Mytilus edulis (Hvilsom, 1983) and Pimephales promelas (Shlueter et al., 1997). Chagnon and Guttman (1989) have shown that in several fish species the enzymatic activity of PGI was significantly reduced by the presence of copper. Direct inhibition of the catalytic activity of PGI by heavy metals was also suggested (Lavie and Nevo, 1982), and individuals with different PGI genotypes have been supposed to be affected by variable degrees of inhibition (Kramer and Newman, 1994). Hvilsom (1983), by means of laboratory experiments, confirmed that copper may act as a selective agent determining shifts in genotypic frequencies at the PGI locus, but hypothesised that factors unrelated to a direct effect on the glucose-6-phosphate-isomerase could contribute in determining the observed changes. Mulvey et al. (1995) suggested that it is difficult to discriminate between adaptations related to specific allozyme genotypes or to variation in closely linked loci (genetic ‘‘hitchhiking’’). Moreover, shifts in genotypic patterns may occur as a consequence of other non-selective processes, such as changes in population size induced by contaminants or other stochastic factors (Belfiore and Anderson, 2001). Genotype-tolerance responses to copper stress observed in H. diversicolor at ALD and PGI loci were consistent among the different sites examined. This strongly reduces the possibility of confounding effects due to non-adaptive processes such as stochastic changes in genotypic frequencies, supporting the model of general adaptive responses to copper stress in H. diversicolor. Previous analyses of the genetic structure of H. diversicolor in the Pialassa lagoons (Virgilio et al., 2003), suggested a possible role of contaminant-induced adaptive responses along a gradient of mercury contamination. The higher frequencies of PGI102/102 described in one of the most contaminated sites of the Pialassa lagoons (Virgilio et al., 2003) could be possibly related to the higher tolerance to copper observed in specimens with PGI102/102 genotype. These results may support the hypothesis by Kramer and Newman (1994) that PGI genotypes act as non-specific markers of acute toxicity. The present study shows the occurrence of genetic based differences in tolerance to copper stress in H. diversicolor. Under laboratory conditions, copper acts as an effective selective agent, promoting differential mortality of individuals with specific PGI and ALD genotypes. The genotype-tolerance responses at PGI and ALD loci suggest that they may be used as markers of genetic responses to heavy metal stress in H. diversicolor.
Acknowledgments J. Anderson, F. Bertozzi, M. Carrera, A. Pasteris and M. Ponti provided excellent assistance at different stages of the study. We wish to thank T. Backeljau and F. Regoli for their critical comments on earlier drafts of the manuscript and F. Bulleri for his valuable suggestions. This paper was written under the EU funded research contract EVK3-CT-2001-00048 (EUMAR).
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varying salinity-copper regimes. Water Air and Soil Pollution 49, 215–230. Ozoh, P.T.E., 1994. The effect of salinity, temperature and time on the accumulation and depuration of copper in ragworm, Hediste diversicolor (O.F. Muller). Environmental Monitoring and Assessment 29, 155–166. Pasteur, N., Pasteur, G., Bonhomme, J., Catalan, J., Britton-Davidian, J., 1988. Practical Isozyme Genetics. Ellis Horwood, Chichester, UK. Patarnello, T., Guinez, R., Battaglia, B., 1991. Effects of pollution on heterozygosity in the barnacle Balanus amphitrite (Cirripedia: Thoracica). Marine Ecology Progress Series 70, 237–243. Schneppenheim, R., Mac Donald, C.M., 1984. Genetic variation and population structure of krill (Euphausia superba) in the Atlantic sector of Antarctic waters and of the Antarctic peninsula. Polar Biology 3, 19–28. Shlueter, M.A., Guttman, S.I., Duan, Y., Oris, J.T., Xiaodong, H., Burton, G.A., 2000. Effects of acute exposure to fluoranthenecontaminated sediment on the survival and genetic variability of fathead minnows (Pimephales promelas). Environmental Toxicology and Chemistry 19, 1011–1018. Shlueter, M.A., Guttman, S.I., Oris, J.T., Bailer, A.J., 1997. Differential survival of fathead minnows, Pimephales promelas, as affected by copper exposure, prior population stress, and allozyme genotypes. Environmental Toxicology and Chemistry 16, 939–947. Troncoso, L., Galleguillos, R., Larrain, A., 2000. Effects of copper on the fitness of the Chilean scallop Argopecten purpuratus (Mollusca: Bivalvia). Hydrobiologia 420, 185–189. Virgilio, M., Baroncini, N., Trombini, C., Abbiati, M., 2003. Relationships between sediments and tissues contamination and allozymic patterns in Hediste diversicolor (Polychaeta: Nereididae) in the Pialassa lagoons (North Adriatic Sea). Oceanologica Acta 26, 85–92.
Allozyme genotypes and tolerance to copper stress in Hediste diversicolor (Polychaeta: Nereididae) Massimiliano Virgilio *, Marco Abbiati Centro Interdipartimentale di Ricerca per le Scienze Ambientali in Ravenna and Dipartimento di Biologia Evoluzionistica e Sperimentale –– University of Bologna, Via S. Alberto 163, I-48100, Ravenna, Italy
Abstract This study analysed the occurrence of genotypic shifts in laboratory populations of Hediste diversicolor (Polychaeta: Nereididae) exposed to copper stress. Specimens of H. diversicolor were collected at three sites, up to 10 km apart, in the estuarine area of the Pialassa lagoons (North Adriatic Sea, Italy) and were used in acute toxicity tests. Specimens were assigned to copper exposure (0.34 mg/l Cu2+) or control conditions. Each combination of Treatment and Site was replicated in two tanks containing 35 specimens of H. diversicolor. The genotypic structure of both dead and survived specimens was analysed by allozyme electrophoresis at six loci (ALD, FH, HBDH LDH, PGI, SDH). Under copper exposure, specimens with the genotypes ALD100/100 and PGI102/102 had significantly lower mortalities than other genotypes Results were consistent across the three sites, suggesting that, under laboratory conditions, effects of copper stress on H. diversicolor is related to individual genotypes at ALD and PGI loci. 2004 Elsevier Ltd. All rights reserved. Keywords: Hediste diversicolor; Toxicity tests; Copper; Tolerance; Selection; Allozymes
1. Introduction Responses of organisms to contaminants can be a continuum of biochemical, physiological or genetic changes, both at population and at community levels (Troncoso et al., 2000). Survival and reproductive capabilities of individuals were shown to be widely affected by their tolerance to contaminants (Grant et al., 1989). Variability in tolerance levels observed in populations exposed to several categories of contaminants has been suggested to have genetic bases (Bryan and Hummerstone, 1971; Gamenick et al., 1998). Contaminants were hypothesised to cause adaptive changes in the genetic structure of populations by means of differential survivorship and reproduction of individuals (Gillespie and *
Corresponding author. Tel.: +39 0544 600300; fax: +39 0544 600411. E-mail address: [email protected] (M. Virgilio). 0025-326X/$ - see front matter 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.marpolbul.2004.06.027
Guttman, 1993). However, due to the complex interactions of evolutionary and physiological processes, it is generally difficult to show a correlation between concentrations of contaminants and adaptive responses of impacted populations (Belfiore and Anderson, 2001). Potentially, other factors, unrelated to tolerance, can alter the genetic structure of impacted populations, e.g. founder effects resulting from severe reductions in size of populations or selection on traits unrelated to tolerance (Bickham et al., 2000). Moreover, tolerance may arise through many other alternative mechanisms, including epistatic interactions (Danzmann et al., 1999) and physiologic acclimation (Kovatch et al., 2000). For these reasons tolerance is not always synonymous of genetic adaptation and is not always inherited through a specific allele or set of alleles (Belfiore and Anderson, 2001). Copper contamination has been supposed to represent a major selective force promoting the occurrence
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of tolerant strains in different species of invertebrates (Lavie and Nevo, 1982) and fishes (Chagnon and Guttman, 1989). Copper, an essential element in metabolic processes, enters aquatic environments from many sources, including wastewater of industrial and agricultural activities, and becomes toxic when water concentrations exceed 0.010 mg/l (Shlueter et al., 1997). Hediste diversicolor (O.F. Mu¨ller) successfully colonises heavily polluted estuarine sediments with copper concentrations up to 4000 lg/g dw (Bryan and Hummerstone, 1971). This species absorbs copper from ingested sediments as well as from solution (Bryan and Hummerstone, 1971) and the occurrence of regulatory and/or excretory capabilities has been hypothesised (Ozoh, 1994). Absence of a true pelagic phase in the life cycle of H. diversicolor has been supposed to have a major role in favouring adaptation to stress, promoting strong selection of juveniles in environments contaminated by heavy metals (Bryan et al., 1987). A steady decline in tolerance to copper was described at increasing distances from heavily polluted areas (Grant et al., 1989; Bryan and Hummerstone, 1971). Tolerance was shown to be neither readily gained nor readily lost under laboratory conditions and the occurrence of adaptation processes, through the development of metal tolerant strains, has been hypothesised (Bryan and Hummerstone, 1971). Laboratory toxicity tests showed that resistance to copper stress in H. diversicolor had a heritable component, providing evidence for the genetic bases of tolerance (Grant et al., 1989). Moreover, analysis of H. diversicolor along a contamination gradient (Virgilio et al., 2003), revealed non-random patterns of variation at six allozyme loci. Patterns of polymorphism appeared to be possibly related to levels of contamination by heavy metals, with major differences occurring at the most impacted sites (Virgilio et al., 2003). In this study we tested hypotheses about relationships between exposure to copper stress and genetic responses in H. diversicolor. The main objectives were (a) to assess if, under laboratory conditions, copper tolerance may be related to differences in genotypic patterns of H. diversicolor and (b) to verify the occurrence of consistent genetic responses among different sites.
2. Materials and methods Genotype-tolerance responses were analysed in populations of H. diversicolor colonising the estuarine area of the Pialassa lagoons (44.4673–44.5293 N, 012.2375– 012.2705 E, European Datum 1950). This area, located along the northern Adriatic coast of Italy, is characterised by the presence of two interconnected lagoons and a river estuary (Fig. 1). A detailed description of the characteristics of the area can be found in Virgilio et al. (2003) and references therein. Average concentra-
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Fig. 1. Estuarine area of the Pialassa lagoons (North Adriatic Sea, Italy). Specimens of Hediste diversicolor were collected at Sites 1, 2 and 3.
tions of copper in sediments of the Pialassa lagoons are two to three times higher than background levels (Miserocchi et al., 1990), with concentrations ranging from 11 to 280 lg/g dw (Anconelli et al., 1980). Replicate ABS tanks (400 · 300 · 80 mm), containing 3 l of 10 psu artificial seawater (Red Sea Salt, Red Sea Fish pHarm, Israel), were used for toxicity tests. Tanks were continuously aerated and maintained at a temperature of 20 C. Worms were acclimatized for 72 h before exposures, no mortality was observed during the acclimation periods. Specimens were not fed during the experiments. Copper was added as CuSO4. Preliminary range finding experiments were conducted in order to determine the target concentrations of copper (96 h, 50% mortality). Based on the results of the range finding experiments, a target concentration of 0.34 mg/l Cu2+ was used to produce a relevant mortality of specimens of H. diversicolor in a 96-h acute toxicity test. Specimens of H. diversicolor, collected in May 2002, were assigned to copper exposure (0.34 mg/l Cu2+) or control conditions. In order to assess the generality of genotype-tolerance responses in populations of H. diversicolor from the estuarine area of the Pialassa lagoons, specimens were sampled at three different sites up to 10 km apart (Fig. 1). At each site, about 140 specimens of H. diversicolor were collected. Two tanks for each combination of treatment and site were used as independent replicates for statistical analyses. To allow an estimation of the genotypic variability within each replicate, 35 specimens of H. diversicolor were randomly assigned to each tank. Dead worms were removed at least every 12 h, rinsed with distilled water and then stored in Eppendorf tubes at 80 C for the genetic analyses. At the end of the 96-h
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period, surviving H. diversicolor specimens were sacrificed and processed using the same procedure. Differences in percentages of mortality were tested by means of a two-way ANOVA with Treatment (0.34 mg/l Cu2+ exposure, control) as fixed factor and Site (1, 2 and 3) as random, orthogonal factor (n = 2). Six allozyme loci showing non-random patterns of variation along a pollution gradient (Virgilio et al., 2003) were analysed by means of cellulose acetate electrophoresis: ALD (aldolase, E.C. 4.1.2.13); FH (fumarate hydratase, E.C. 4.2.1.2); HBDH (3-hydroxybutyrate dehydrogenase, E.C. 1.1.1.30); LDH (lactate dehydrogenase, E.C. 1.1.1.27); PGI (glucose-6-phosphate isomerase, E.C. 5.3.1.9) and SDH (sorbitol dehydrogenase, E.C. 1.1.1.14). For each specimen of H. diversicolor, about 100 mg of tissue from the central setigers was homogenised with 100 ll of extracting buffer (Tris–HCl 0.05 M pH 8, TritonX100 1:1000), subjected to electrophoresis and stained following the procedures reported in Pasteur et al. (1988) with slight modifications. Tris– EDTA–Maleic acid (pH 7.8) electrode buffer (Schneppenheim and Mac Donald, 1984) was used in 1:1 and 1:2 dilutions. For each locus the most common allele in the reference population (Site 1) was designated as allele 100. Slower and faster bands on the zymograms, representing other alleles, were given lower and higher numbers corresponding to their relative mobility. Linkage disequilibrium among genotypes at pairs of loci in each site was calculated using a log-likelihood ratio G-statistic, as implemented by the computer package FSTAT 1.2 (Goudet, 1995). Multiple tests were adjusted by the sequential Bonferroni correction (Hochberg, 1988). For each of the replicate tanks exposed to copper (0.34 mg/l Cu2+), observed heterozygosity (e.g. the observed proportion of heterozygotes at each locus) was calculated for both dead and survived specimens after 96 h. Differences in observed heterozygosity were tested by means of a two-way ANOVA with Endpoint (dead, survived) as fixed factor and Site (1, 2 and 3) as random, orthogonal factor (n = 2). For each of the replicate
tanks, the mortality ratios associated to the observed genotypes (number of dead/number of survived specimens with the same genotype) were calculated after 96 h. At each locus, differences in mortality ratios were tested by means of a three-way ANOVA with Site (1, 2 and 3) as random factor, Treatment (0.34 mg/l Cu2+, control) and Genotype as fixed, orthogonal factors (n = 2). Assumption of homogeneity of variances for percentages of mortality, heterozygosity values and mortality ratios was tested by means of CochranÕs C-test. In order to identify possible trends in percentages of mortality, heterozygosity levels and mortality ratios, the Student–Newman–Keuls (SNK) test was used for a posteriori multiple comparison of means.
3. Results Average percentages of mortality over the three sites were 3.0% (S.E. = 1.1) in controls and 41.6% (S.E. = 4.2) in treatments exposed to 0.34 mg/l Cu2+. ANOVA and SNK tests (Table 1) did not reveal differences in percentages of mortality among controls while, in copper exposures, specimens of H. diversicolor from Site 2 showed a significantly higher percentage of mortality (m% = 54.4, S.E. = 1.5) compared to Site 1 (m% = 33.8, S.E. = 0.5) and Site 3 (m% = 36.7, S.E. = 3.3). No linkage disequilibrium was observed out of 58 pairwise comparisons of loci. Average observed heterozygosity values (Hobs) were: 0.149 (S.E. = 0.029) at ALD; 0.073 (S.E. = 0.026) at FH; 0.003 (S.E. = 0.003) at HBDH; 0.100 (S.E. = 0.012) at LDH; 0.303 (S.E. = 0.055) at PGI and 0.084 (S.E. = 0.030) at SDH. In specimens exposed to copper, ANOVA on observed heterozygosity at polymorphic loci ALD, FH, PGI and SDH (Fig. 2) did not reveal significant differences, either between dead and surviving specimens or between sites. Yet, ANOVA showed a significant Site · Endpoint interaction at LDH locus, although SNK did not suggest any alternative to the null hypothesis (Table 2). ANOVA on
Table 1 ANOVA and SNK test for the effects of Treatment (0.34 mg/l Cu2+ exposure, control) and Site (1, 2 and 3) on percentages of mortality of Hediste diversicolor Source of variation
df
MS
F
P
Treatment = T Site = S T·S Residual CochranÕs C-test (C = 0.401 p > 0.05) Transformation (None)
1 2 2 6
4466.02 152.75 100.78 9.33
44.31 16.37 10.80
* ** *
SNK test: Treatment · Site 0.34 mg/l Cu2+: Control: *p
< 0.05;
**p
< 0.01.
Site 1 = Site 3 < Site 2 Site 3 = Site 1 = Site 2
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< 0.05; n.s., not significant. *p
Dead: Site 1 = Site 2 = Site 3 Survived: Site 1 = Site 2 = Site 3
SNK test: Site · Endpoint LDH
2 1 2 6 Site = S Endpoint = E S·E Residual CochranÕs C-test Transformation
0.023 2.49 0.014 3.57 0.004 0.42 0.009 C = 0.559 p > 0.05 None
n.s. n.s. n.s.
0.001 0.52 n.s. 0.000 0.03 n.s. 0.003 1.48 n.s. 0.002 C = 0.639 p > 0.05 None
0.011 2.11 n.s. 0.014 0.49 n.s. 0.028 5.61 * 0.005 C = 0.313 p > 0.05 None
0.006 0.62 n.s. 0.005 2.54 n.s. 0.002 0.22 n.s. 0.009 C = 0.423 p > 0.05 None
0.032 1.93 n.s. 0.001 1.00 n.s. 0.001 0.07 n.s. 0.016 C = 0.679 p > 0.05 Square root
Residual S·E Residual
Denominator for F P F MS
SDH
P F MS
PGI
P F MS
LDH
P MS
F FH
F
P MS
It is often difficult to generalise about the toxicity of heavy metals in aquatic organisms because of the active role that organisms play in regulating bioaccumulation/ detoxification processes and of the influence of physical/ chemical variables such as salinity and temperature (Ozoh, 1994). Several processes were suggested to cause differences in tolerance to copper stress among populations of Hediste diversicolor. Variations in permeability of the body surface could affect accumulation and detoxification processes (Bryan and Hummerstone, 1971; Bryan et al., 1987). Copper accumulation in
ALD
4. Discussion
df
mortality ratios (Table 3) showed a significant Treatment · Genotype interaction at ALD and PGI loci. SNK tests revealed that, under copper stress, mortality ratios associated to genotype ALD100/100 were significantly lower than those associated to genotypes ALD100/102 and ALD102/102, while no differences were observed under control conditions (Fig. 3). Similarly, under copper stress, mortality ratios associated to genotype PGI102/102 were significantly lower than those associated to genotypes PGI100/102 and PGI102/102, while no differences were observed under control conditions (Fig. 4). At PGI locus, there was also a significant Site · Treatment interaction, due to the higher mortality ratios in Site 2. Genetic variability at loci HBDH, FH and SDH was very low, therefore ANOVA on effects of copper stress on heterozygosity and on mortality ratios was not performed.
Source of variation
Fig. 2. Hediste diversicolor. Observed heterozygosity (Hobs) of dead and survived specimens after 96-h exposure to 0.34 mg/l Cu2+. Results are shown at ALD, FH, LDH, PGI and SDH loci. Sites as in Fig. 1.
Table 2 Hediste diversicolor. ANOVA and SNK test for the effects of Site (1, 2 and 3) and Endpoint (dead, survived) on observed heterozygosity values at loci ALD, FH, LDH, PGI and SDH in specimens exposed to 0.34 mg/l Cu2+
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Source of variation
PGI
ALD
df Genotype = G Site = S Treatment = T G·S G·T S·T G·S·T Residual CochranÕs C-test Transformation
MS
2 0.211 2 0.297 1 2.351 4 0.013 2 0.157 2 0.260 4 0.011 18 0.069 C = 0.4037 p > 0.05 None
P
df
15.97 4.31 9.04 0.19 14.58 3.78 0.16
* * n.s. n.s. * * n.s.
2 0.967 2 2.033 1 8.920 4 0.169 2 0.829 2 1.159 4 0.019 18 0.782 C = 0.435 p > 0.05 None
SNK test: Genotype · Treatment
PGI 2+
0.34 mg/l Cu : Control:
100/102 = 100/100 > 102/102 100/102 = 100/100 = 102/102
PGI
SNK test: Site · Treatment 2+
0.34 mg/l Cu : Control:
LDH
F
MS
**p
P
df
MS
5.74 2.6 7.69 0.22 44.35 1.48 0.02
n.s. n.s. n.s. n.s. ** n.s. n.s.
1 2 1 2 1 2 2 12 C = 0.597 None
0.019 0.01 0.713 0.64 0.109 1.57 1.923 1.72 0.032 0.05 0.070 0.06 0.646 0.58 1.117 0.01 < p < 0.05
SNK test: Genotype · Treatment
ALD 2+
0.34 mg/l Cu : Control:
Site 2 > Site 3 = Site 1 Site 2 = Site 1 = Site 3
Results are shown for PGI, ALD, and LDH loci, *p < 0.05;
F
< 0.01; n.s. = not significant.
102/102 = 100/102 > 100/100 102/102 = 100/100 = 100/102
F
P
Denominator for F
n.s. n.s. n.s. n.s. n.s. n.s. n.s.
G·S Residual S·T Residual G·S·T Residual Residual
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Table 3 Hediste diversicolor. ANOVA and SNK test for the effects of Genotype, Site and Treatment on mortality ratios (number of dead/number of survived specimens with the same genotype)
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0.34 mg/l Cu
4
2+
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ALD
PGI control
0.34 mg/l Cu2+
control
Site 1
Site 1
2 2 0
1 0
4
1 0 2 1 0
4
2 Mean
0
2
1
genotype
100/100
100/102
102/102
100/100
100/102
102/102
100/100
100/102
102/102
100/100
0 100/102
0
102/102
Mean
2
Site 2
mortality ratio
4
Site 3
Site 2
0
Site 3
mortality ratio
2 2
genotype
Fig. 3. Hediste diversicolor. Mortality ratios (number of dead/number of survived specimens with the same genotype) associated to locus ALD. Sites as in Fig. 1.
Fig. 4. Hediste diversicolor. Mortality ratios (number of dead/number of survived specimens with the same genotype) associated to locus PGI. Sites as in Fig. 1.
H. diversicolor has been shown to occur more readily at low salinities (Ozoh, 1990), when the uptake of divalent ions may be favoured both by lower competition from calcium and magnesium ions and potential differences across the body surface (Bryan et al., 1987). Higher temperatures have also been shown to increase the accumulation of copper and were supposed to influence changes of heavy metals tolerances (Ozoh, 1994). In this study, different percentages of mortality were observed among sites after exposure to 0.34 mg/l Cu2+, suggesting the occurrence of differently tolerant strains of H. diversicolor. Differences in local environmental conditions at study sites (salinity variations, temperature ranges, levels of contamination, etc.) may have enhanced the selection/acclimation of physiologically different strains of H. diversicolor. Heterozygous individuals at allozyme loci may have lower metabolic requirements and higher efficiency in several metabolic processes (Koehn, 1989). Different studies showed relationships between single- or multilocus heterozygosity and higher tolerances to heavy metal stressors (Diamond et al., 1991; Troncoso et al., 2000). Authors have suggested that heterozygotes, producing different forms of the same metabolic enzyme, may be better buffered against environmental fluctua-
tions (Diamond et al., 1991). Under the experimental conditions used in this study, tolerance to copper stress in H. diversicolor was not related to levels of heterozygosity. Conversely, exposure to copper resulted in higher survivorship of specimens of H. diversicolor owing specific allozyme genotypes. After copper exposure, mortality ratios associated to ALD100/100 and PGI102/102 genotypes were sensibly lower, supporting the model that higher tolerance to copper stress is related to the occurrence of these genotypes. Results are in agreement with other studies showing that in species such as Hyalella azteca (Duan et al., 2001), Pimephales promelas (Shlueter et al., 1997, 2000), Gambusia affinis (Newman et al., 1989) and G. holbrooki (Diamond et al., 1991), tolerance was not associated to heterozygosity levels, but to specific genotypes. Glucose-6-phosphate-isomerase (PGI) has been widely studied, both for its role in the metabolic processes and for its high levels of polymorphism (Troncoso et al., 2000). Previous studies on genetic adaptation to contaminants showed that PGI genotypes are frequently associated to tolerance responses to specific stressors, such as PAH (Shlueter et al., 2000), cadmium (Lavie and Nevo, 1986), mercury (Mulvey et al., 1995), zinc (Lavie and Nevo, 1982) or general levels of pollution (Patarnello et al., 1991).
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Copper exposure resulted in differential survivorship of individuals with different PGI genotypes in species such as Monodonta turbinata, M. turbiformis (Lavie and Nevo, 1982), Mytilus edulis (Hvilsom, 1983) and Pimephales promelas (Shlueter et al., 1997). Chagnon and Guttman (1989) have shown that in several fish species the enzymatic activity of PGI was significantly reduced by the presence of copper. Direct inhibition of the catalytic activity of PGI by heavy metals was also suggested (Lavie and Nevo, 1982), and individuals with different PGI genotypes have been supposed to be affected by variable degrees of inhibition (Kramer and Newman, 1994). Hvilsom (1983), by means of laboratory experiments, confirmed that copper may act as a selective agent determining shifts in genotypic frequencies at the PGI locus, but hypothesised that factors unrelated to a direct effect on the glucose-6-phosphate-isomerase could contribute in determining the observed changes. Mulvey et al. (1995) suggested that it is difficult to discriminate between adaptations related to specific allozyme genotypes or to variation in closely linked loci (genetic ‘‘hitchhiking’’). Moreover, shifts in genotypic patterns may occur as a consequence of other non-selective processes, such as changes in population size induced by contaminants or other stochastic factors (Belfiore and Anderson, 2001). Genotype-tolerance responses to copper stress observed in H. diversicolor at ALD and PGI loci were consistent among the different sites examined. This strongly reduces the possibility of confounding effects due to non-adaptive processes such as stochastic changes in genotypic frequencies, supporting the model of general adaptive responses to copper stress in H. diversicolor. Previous analyses of the genetic structure of H. diversicolor in the Pialassa lagoons (Virgilio et al., 2003), suggested a possible role of contaminant-induced adaptive responses along a gradient of mercury contamination. The higher frequencies of PGI102/102 described in one of the most contaminated sites of the Pialassa lagoons (Virgilio et al., 2003) could be possibly related to the higher tolerance to copper observed in specimens with PGI102/102 genotype. These results may support the hypothesis by Kramer and Newman (1994) that PGI genotypes act as non-specific markers of acute toxicity. The present study shows the occurrence of genetic based differences in tolerance to copper stress in H. diversicolor. Under laboratory conditions, copper acts as an effective selective agent, promoting differential mortality of individuals with specific PGI and ALD genotypes. The genotype-tolerance responses at PGI and ALD loci suggest that they may be used as markers of genetic responses to heavy metal stress in H. diversicolor.
Acknowledgments J. Anderson, F. Bertozzi, M. Carrera, A. Pasteris and M. Ponti provided excellent assistance at different stages of the study. We wish to thank T. Backeljau and F. Regoli for their critical comments on earlier drafts of the manuscript and F. Bulleri for his valuable suggestions. This paper was written under the EU funded research contract EVK3-CT-2001-00048 (EUMAR).
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