- Email: [email protected]
Cytogenetics of Anodonta cygnea (Mollusca: Bivalvia) as possible indicator of environmental adversity
Estuarine, Coastal and Shelf Science 80 (2008) 303–306
Contents lists available at ScienceDirect
Estuarine, Coastal and Shelf Science journal homepage: www.elsevier.com/locate/ecss
Short Communication
Cytogenetics of Anodonta cygnea (Mollusca: Bivalvia) as possible indicator of environmental adversity J. Carrilho a, *, A. Leita˜o c, C. Vicente b, I. Malheiro a a
Laboratory of Cytogenetics, ICBAS – Instituto de Cieˆncias Biome´dicas de Abel Salazar, Universidade do Porto Largo Prof. Abel Salazar, 2, 4099-003 Porto, Portugal Population Studies Department, ICBAS – Instituto de Cieˆncias Biome´dicas de Abel Salazar, Universidade do Porto Largo Prof. Abel Salazar, 2, 4099-003 Porto, Portugal c ´gicos (INRB, I.P.)/L – IPIMAR, Avenida 5 de Outubro s/n, P-8700-305 Olha ˜o, Portugal Instituto Nacional de Recursos Biolo b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 June 2008 Accepted 23 July 2008 Available online 31 July 2008
Anodonta cygnea is a freshwater clam, belonging to the Unionidae family, which can be found in rivers and lagoons all over Europe and Northern America. As they appear as important case studies for ecological damage assessments, the various species of the Unionidae family have been submitted to a sort of recent studies on their chromosomal or cytogenetic status. In this study we confirmed the diploid chromosome number of 2n ¼ 38 for this species, and established for the first time the karyotype, which comprised six metacentric, 12 submetacentric and one subtelocentric chromosome pairs. We also found a high percentage of cells with an abnormal number of chromosomes. Considering that karyotype disturbances in Unionids have been previously related with exposure to chemicals, either natural or produced by human activity, we determined the aneuploidy index for our population. The aneuploidy index is an excellent marker for pollutant presence/effect. The animals acclimatized in tap water and in natural water from the lake where the individuals were collected showed different levels of aneuploidy. The higher values were found in tap water. Chromosome analysis techniques seem a suitable tool to study the impact of contaminants referred above, and making A. cygnea a suitable organism for assessment of an eugenic damage in aquatic systems. On the other hand, our results also point out to the importance of doing the acclimatizing process of the collected animals in their own natural water. Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Anodonta cygnea chromosomes freshwater molluscs environmental factors genetic abnormalities karyotype Lagoa de Mira Portugal
1. Introduction Among freshwater animals, clams (Unionacea) are the most common and widespread species (Graf and Cummings, 2007). The chromosome number within Unionidae is known for 27 species, with most of them having 38 chromosomes (for a review see Nakamura, 1985; Thiriot-Quie´vreux, 2002). A renewed interest in the chromosomal or cytogenetic status of various species of the Unionidae family has been generated by their importance as ecological damage case studies (Barsiene, 1994; Barsiene and Lovejoy, 2000). In bivalves, exposure to chemicals, either natural or produced by human activity, may result, namely, in karyotype disturbances such as aneuploidy (the occurrence of one or more extra or missing chromosomes leading to an unbalanced chromosome complement, or, any chromosome number that is not an exact multiple of the haploid number) (e.g. Melnychenko, 2002; Bouilly et al., 2004; Barsiene et al., 2006). Drinking water is contaminated with a large number of organic compounds, since the aquifers where they come from are a sink for
* Corresponding author. E-mail address: [email protected] (J. Carrilho). 0272-7714/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.ecss.2008.07.019
many natural and anthropogenically derived chemicals, namely from the agricultural, pharmaceutical, oil and gas industries (Andrade et al., 2008). Studies of genotoxicity in drinking water have already been performed in Anodonta sp. (Scarpato et al., 1990; Barsiene et al., 2006). In the above mentioned studies, micronuclei were used as biomarkers. They are indicators of aneuploidy as they appear as smaller secondary nuclei in the cell, result of the delay of a chromosome fragment or a whole chromosome during the anaphase (Heddle et al., 1991). The aim of this work was, to describe for the first time the karyotype of Anodonta cygnea, and also to demonstrate how cytogenetic studies, in this particular case, aneuploidy scoring, can be highly useful tools in the assessment of environmental-individual relationship, emphasizing the chromosomal damage that can arise from the process of acclimation in tap water. 2. Material and methods For the standard karyotype 50 specimens of A. cygnea were collected from natural populations in Lagoa de Mira, Portugal (40 270 N, 8 470 W) (Fig. 1). To obtain mitotic chromosomes, animals were incubated for 6–7 h with 0.1% colchicine in fresh water. The gills were then
304
J. Carrilho et al. / Estuarine, Coastal and Shelf Science 80 (2008) 303–306
3. Results The diploid number consists of 38 chromosomes with an NF ¼ 76. The relative length of the chromosomes ranged between 3.72 and 7.34 and the karyotype (Fig. 2 and Table 1) was constituted by six pairs of metacentric chromosomes (m), 11 pairs of submetacentric chromosomes (sm) and one pair of subtelocentric chromosomes (st). Our aneuploidy counts showed that cell hypodiploidy (cells consisting of one or more chromosome less than in diploid sets, see Fig. 3a) was present in 47.1% of the cells from animals acclimated in tap water and in 9.6% of those from animals acclimated in water from the lake. Cell hyperdiploidy (cells consisting of one or more chromosomes more than in diploid sets, see Fig. 3b) was present in 5.9% of the cells from animals acclimated in tap water and in 5.5% of those from animals acclimated in water from the lake. Cell polyploidy (multiple diploid sets in the same cell, see Fig. 3c) wasn’t detected in animals acclimated in tap water, and was found in 4.1% of the cells from animals acclimated in lake water. Fig. 1. Map of the Iberian Peninsula, showing the location of the Lagoa de Mira.
4. Discussion dissected out and treated for 90 min in distilled water. The material was then fixed in a freshly prepared solution of absolute ethanol and acetic acid (3:1), with three changes of 20 min each. Chromosome spreads were obtained through the air-drying technique of Thiriot-Quie´vreux and Ayraud (1982). Slides were directly coloured with Giemsa (4%, pH 6.8) for 8 min. From the about 130 metaphases observed in 30 animals, in which we could confirm the diploid number of 2n ¼ 38, we selected 67 intact and well-spread ones for chromosome analyses and karyotype determination. This was performed using a Nikon Ecclipse E400 microscope with an F-View II digital camera, with Cell ID software associated. Karyotype formula was determined after statistical analysis of chromosome measurements: relative lengths and centromeric indexes. Chromosome nomenclature follows that of Levan et al. (1964). To perform the aneuploidy counts, the remaining animals were divided into 2 groups of nine animals each. The first group was acclimated in water from the natural collection site, and the second group was placed in drinking tap water. Acclimation period was of 2 weeks. The temperature and feeding protocol were the same for both groups. Slides were executed in the same way as referred before and stained with Giemsa. Chromosome number was determined for 100 metaphases and aneuploidy indexes determined for each group, by calculating the percent of cells with abnormal chromosome number in the total pool of cells analyzed.
The diploid number of 2n ¼ 38 for A. cygnea described here (Fig. 2) is coincident with that reported for this species and other Anodonta sp. (A. grandis, A. piscinalis, A. subcircularis, A. woodiana) (Nakamura, 1985; Barsiene, 1994; Woznicki, 2004; Woznicki and Jankun, 2004). The fundamental chromosome arm number (NF) reported in the present paper is similar to that described for four other Unionidae species, and equalled 76 (only bi-armed chromosomes were found) (Nakamura, 1985). The relative length and morphology of chromosomes seems to be similar to A. anatine. In regard to A. woodiana, only the morphology of chromosomes was different (Woznicki, 2004; Woznicki and Jankun, 2004). The main differences between these two karyotypes were the number of submetacentric chromosomes in A. cygnea (12 pairs), which was higher than in A. woodiana (five pairs), and the presence of one pair of subtelocentric chromosomes in A. cygnea. Bivalves are organisms that can accumulate in their tissues several kinds of pollutants. Even in sublethal concentrations, these chemicals may have genotoxic effects that namely translate into aneuploidy (Anderson et al., 1994). These effects are clearly alerting symptoms of environmental adversity (Sokolowski et al., 2004). In our study, the result obtained for the total abnormal metaphase index (aneuploidy and polyploidy) in the lake water was of 19.2% (Fig. 4). In comparison to the reported levels of natural aneuploidy in Anodonta cygnea (Barsiene and Bucinskiene, 2001; Barsiene et al., 2002) we find that these are already above what was expected for an unpolluted site. Thus, we suspect that our collection
Fig. 2. Giemsa-stained diploid metaphase and respective karyotype of Anodonta cygnea.
J. Carrilho et al. / Estuarine, Coastal and Shelf Science 80 (2008) 303–306 Table 1 Chromosome measurements and classification Chromosome pair number
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Relative length
Arm ratio
Centromeric index
Mean
SD
Mean
SD
Mean
SD
6.72 5.98 5.33 4.62 4.15 3.72 7.34 6.41 6.10 5.89 5.51 5.39 5.30 5.08 4.96 4.62 4.31 3.78 4.80
0.88 0.75 0.60 0.55 0.51 0.42 1.03 0.80 0.73 0.71 0.71 0.65 0.61 0.61 0.63 0.53 0.49 0.41 0.58
1.26 1.31 1.26 1.29 1.35 1.21 1.86 2.09 2.07 2.14 2.38 2.10 2.13 2.10 2.21 2.24 2.03 1.91 4.11
0.15 0.21 0.19 0.19 0.18 0.12 0.23 0.25 0.36 0.33 0.31 0.26 0.26 0.44 0.44 0.41 0.28 0.32 1.11
44.48 43.66 44.48 43.95 42.82 45.29 35.13 32.54 33.02 32.14 29.81 32.45 32.17 32.87 32.24 31.38 33.23 35.13 20.32
3.03 3.67 3.61 3.45 3.29 2.40 2.81 2.64 3.68 3.26 2.76 2.56 2.64 5.07 4.42 4.04 2.69 4.17 3.72
Classification
m m m m m m sm sm sm sm sm sm sm sm sm sm sm sm st
305
site was already contaminated, supposedly by the drains of surrounding agricultural fields and residential areas. Nevertheless, the diploid number in the majority of the cells was 38 (similar to that described in other works, as mentioned above). Therefore, we considered the data from this population safe to establish the standard karyotype of this species, based on the centromeric index, the arm ratio and the relative length of the chromosome (Fig. 2 and Table 1). Our studies in Anodonta cygnea, acclimatized in drinking tap water and in natural water from the lake where the individuals were collected, showed significant different indexes of aneuploidy (with special emphasis to hypodiploidy) between individuals in either rearing conditions, with higher values being found in tap water. These results are to be expected if we take in consideration that, drinking tap water, is submitted to a series of treatments before distribution. These include chlorination, chloramination and ozonation. All of these are introducing potential aneugenic compounds in the water (Monarca et al., 2004). Our findings are in agreement with what was described by Scarpato et al. (1990), in this same species, using micronucleus assays. Our study provides, nevertheless, a more refined scenario of the changes in genetic information involved in these processes of
Fig. 3. (a) Hypodiploid metaphase 2n ¼ 35 and respective karyotype. (b) Hyperdiploid metaphase 2n ¼ 42 and respective karyotype. (c) Polyploid metaphase 4n ¼ 76 and respective karyotype.
306
J. Carrilho et al. / Estuarine, Coastal and Shelf Science 80 (2008) 303–306
tap water
a
Acknowledgements
5.9%
The authors acknowledge Prof. Doutora Beatriz Porto for all the assistance and advice, Prof. Doutor Jorge Machado for providing the algal suspension and Sr. Correia for the help in the collection of the animals.
47.1%
References
47% diploid
b
hypodiploid
hyperdiploid
lake water 5.5%
4.1%
9.6%
80.8% diploid
hypodiploid
hyperdiploid
poliploid
Fig. 4. Amount of each type of cell in tap water (a) and in lake water (b).
toxicity. Namely, we can show increases in chromosome number, otherwise undetected by micronuclei testing, and also determine just how much of the chromosome is lost to the micronuclei.
5. Conclusions In conclusion, we can suggest that: (1) aneugenic agents are present in the water from the Lagoa de Mira; (2) there is a higher aneugenic activity in our laboratory tap water, when compared to the lake water; (3) chromosome analysis techniques are a useful tool to study the impact of contaminants referred above making Anodonta cygnea a suitable organism for assessment of cytogenetic damage in aquatic systems. On the other hand, our results also point out to the importance of doing the acclimatizing process of the collected animals in their own natural water to assure the homeostasis of the animal, and consequently avoid systematic bias in future results.
Anderson, S., Sadinski, W., Shugart, L., Brussard, P., Depledge, M., Ford, T., Hose, E., Stegeman, J., Suk, W., Wirgin, I., Wogan, G., 1994. Genetic and molecular ecotoxicology: a research framework. Environmental Health Perspectives 101, 3–8. Andrade, E.M., Pala´cio, H.A.Q., Souza, I.M., Lea˜o, R.A.O., Guerreiro, M.J., 2008. Land use effects in groundwater composition of an alluvial aquifer (Trussu River, Brazil) by multivariate techniques. Environmental Research 106, 170–177. Barsiene, J., 1994. Chromosome set changes in molluscs from highly polluted habitats. In: Beaumont, A.R. (Ed.), Genetics and Evolution of Aquatic Organisms. Chapman & Hall, London, pp. 434–446. Barsiene, J., Lovejoy, D.B., 2000. Environmental genotoxicity in Klaipeda Port Area. International Review of Hydrobiology 85, 663–672. Barsiene, J., Bucinskiene, R., 2001. Cytogenetic damage in the tissues of indigenous and transferred molluscs. Acta Zoologica Lituanica 11, 102–109. Barsiene, J., Bucinskiene, R., Joksas, K., 2002. Cytogenetic damage and heavy metal bioaccumulation in molluscs inhabiting different sites of the Neris River. Ekologija 2, 52–57. Barsiene, J., Andreikenaite, L., Rybakovas, A., 2006. Cytogenetic damage in perch (Perca fluviatilis L.) and Duck mussel (Anodonta anatina L.) exposed to crude oil. Ekologija 1, 25–31. Bouilly, K., McCombie, H., Leita˜o, A., Lape`gue, S., 2004. Persistence of atrazine impact on aneuploidy in Pacific oysters, Crassostrea gigas. Marine Biology 145, 699–705. Graf, D., Cummings, K., 2007. Review of the systematics and global diversity of freshwater mussel species (Bivalvia: Unionoida). Journal of Molluscan Studies 73, 291–314. Heddle, J.A., Cimino, M.C., Hayashi, M., Romagna, F., Shelby, M.D., Tucker, J.D., Vanparys, Ph., MacGregor, P.T., 1991. Micronuclei as an index of cytogenetic damage: past, present, and future. Environmental and Molecular Mutagenesis 8, 277–291. Levan, A., Fredga, K., Sandberg, A.A., 1964. Nomenclature for centromere position in chromosomes. Hereditas 52, 201–220. Melnychenko, R.K., 2002. About polyploidy and aneuploidy in cells of the family Unionidae (Mollusca, Bivalvia) in the area of Ukraine. Vestnik Zoologii 36, 21–26. Monarca, S., Zani, C., Richardson, S.D., Thruston Jr., A.D., Moretti, M., Feretti, D., Villarini, M., 2004. A new approach to evaluating the toxicity and genotoxicity of disinfected drinking water. Water Research 38, 3809–3819. Nakamura, H.K., 1985. A review of molluscan cytogenetic information based on the CISMOCH-computerized index system for molluscan chromosomes. Bivalvia, Polyplacophora and Cephalopoda. Venus 44, 193–225. Scarpato, R., Migliore, L., Barale, R., 1990. The micronucleus assay in Anodonta cygnea for the detection of drinking water mutagenicity. Mutation Research 245, 231–237. Sokolowski, A., Wolowicz, M., Hummel, H., Smolarz-Go´rska, K., Fichet, D., Radenac, G., Thiriot-Quie´vreux, C., Namiesnik, J., 2004. Abnormal features of Macoma balthica (Bivalvia) in the Baltic sea: alerting symptoms of environmental adversity? Marine Pollution Bulletin 49, 17–22. Thiriot-Quie´vreux, C., Ayraud, N., 1982. Les caryotipes de quelques espe`ces de Bivalves e Gaste´ropodes marins. Marine Biology 70, 165–172. Thiriot-Quie´vreux, C., 2002. Review of the literature on bivalve cytogenetics in the last ten years. Cahiers de Biologie Marine 43, 17–26. Woznicki, P., 2004. Chromosomes of the Chinese mussel Anodonta woodiana (Lea 1834) from the heated Konin Lakes system in Poland. Malacologia 46, 205–210. Woznicki, P., Jankun, M., 2004. Chromosome study of Anodonta anatine (L., 1758) (Bivalvia, Unionidae). Folia Biologica (Krakow) 52, 171–174.
Contents lists available at ScienceDirect
Estuarine, Coastal and Shelf Science journal homepage: www.elsevier.com/locate/ecss
Short Communication
Cytogenetics of Anodonta cygnea (Mollusca: Bivalvia) as possible indicator of environmental adversity J. Carrilho a, *, A. Leita˜o c, C. Vicente b, I. Malheiro a a
Laboratory of Cytogenetics, ICBAS – Instituto de Cieˆncias Biome´dicas de Abel Salazar, Universidade do Porto Largo Prof. Abel Salazar, 2, 4099-003 Porto, Portugal Population Studies Department, ICBAS – Instituto de Cieˆncias Biome´dicas de Abel Salazar, Universidade do Porto Largo Prof. Abel Salazar, 2, 4099-003 Porto, Portugal c ´gicos (INRB, I.P.)/L – IPIMAR, Avenida 5 de Outubro s/n, P-8700-305 Olha ˜o, Portugal Instituto Nacional de Recursos Biolo b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 June 2008 Accepted 23 July 2008 Available online 31 July 2008
Anodonta cygnea is a freshwater clam, belonging to the Unionidae family, which can be found in rivers and lagoons all over Europe and Northern America. As they appear as important case studies for ecological damage assessments, the various species of the Unionidae family have been submitted to a sort of recent studies on their chromosomal or cytogenetic status. In this study we confirmed the diploid chromosome number of 2n ¼ 38 for this species, and established for the first time the karyotype, which comprised six metacentric, 12 submetacentric and one subtelocentric chromosome pairs. We also found a high percentage of cells with an abnormal number of chromosomes. Considering that karyotype disturbances in Unionids have been previously related with exposure to chemicals, either natural or produced by human activity, we determined the aneuploidy index for our population. The aneuploidy index is an excellent marker for pollutant presence/effect. The animals acclimatized in tap water and in natural water from the lake where the individuals were collected showed different levels of aneuploidy. The higher values were found in tap water. Chromosome analysis techniques seem a suitable tool to study the impact of contaminants referred above, and making A. cygnea a suitable organism for assessment of an eugenic damage in aquatic systems. On the other hand, our results also point out to the importance of doing the acclimatizing process of the collected animals in their own natural water. Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Anodonta cygnea chromosomes freshwater molluscs environmental factors genetic abnormalities karyotype Lagoa de Mira Portugal
1. Introduction Among freshwater animals, clams (Unionacea) are the most common and widespread species (Graf and Cummings, 2007). The chromosome number within Unionidae is known for 27 species, with most of them having 38 chromosomes (for a review see Nakamura, 1985; Thiriot-Quie´vreux, 2002). A renewed interest in the chromosomal or cytogenetic status of various species of the Unionidae family has been generated by their importance as ecological damage case studies (Barsiene, 1994; Barsiene and Lovejoy, 2000). In bivalves, exposure to chemicals, either natural or produced by human activity, may result, namely, in karyotype disturbances such as aneuploidy (the occurrence of one or more extra or missing chromosomes leading to an unbalanced chromosome complement, or, any chromosome number that is not an exact multiple of the haploid number) (e.g. Melnychenko, 2002; Bouilly et al., 2004; Barsiene et al., 2006). Drinking water is contaminated with a large number of organic compounds, since the aquifers where they come from are a sink for
* Corresponding author. E-mail address: [email protected] (J. Carrilho). 0272-7714/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.ecss.2008.07.019
many natural and anthropogenically derived chemicals, namely from the agricultural, pharmaceutical, oil and gas industries (Andrade et al., 2008). Studies of genotoxicity in drinking water have already been performed in Anodonta sp. (Scarpato et al., 1990; Barsiene et al., 2006). In the above mentioned studies, micronuclei were used as biomarkers. They are indicators of aneuploidy as they appear as smaller secondary nuclei in the cell, result of the delay of a chromosome fragment or a whole chromosome during the anaphase (Heddle et al., 1991). The aim of this work was, to describe for the first time the karyotype of Anodonta cygnea, and also to demonstrate how cytogenetic studies, in this particular case, aneuploidy scoring, can be highly useful tools in the assessment of environmental-individual relationship, emphasizing the chromosomal damage that can arise from the process of acclimation in tap water. 2. Material and methods For the standard karyotype 50 specimens of A. cygnea were collected from natural populations in Lagoa de Mira, Portugal (40 270 N, 8 470 W) (Fig. 1). To obtain mitotic chromosomes, animals were incubated for 6–7 h with 0.1% colchicine in fresh water. The gills were then
304
J. Carrilho et al. / Estuarine, Coastal and Shelf Science 80 (2008) 303–306
3. Results The diploid number consists of 38 chromosomes with an NF ¼ 76. The relative length of the chromosomes ranged between 3.72 and 7.34 and the karyotype (Fig. 2 and Table 1) was constituted by six pairs of metacentric chromosomes (m), 11 pairs of submetacentric chromosomes (sm) and one pair of subtelocentric chromosomes (st). Our aneuploidy counts showed that cell hypodiploidy (cells consisting of one or more chromosome less than in diploid sets, see Fig. 3a) was present in 47.1% of the cells from animals acclimated in tap water and in 9.6% of those from animals acclimated in water from the lake. Cell hyperdiploidy (cells consisting of one or more chromosomes more than in diploid sets, see Fig. 3b) was present in 5.9% of the cells from animals acclimated in tap water and in 5.5% of those from animals acclimated in water from the lake. Cell polyploidy (multiple diploid sets in the same cell, see Fig. 3c) wasn’t detected in animals acclimated in tap water, and was found in 4.1% of the cells from animals acclimated in lake water. Fig. 1. Map of the Iberian Peninsula, showing the location of the Lagoa de Mira.
4. Discussion dissected out and treated for 90 min in distilled water. The material was then fixed in a freshly prepared solution of absolute ethanol and acetic acid (3:1), with three changes of 20 min each. Chromosome spreads were obtained through the air-drying technique of Thiriot-Quie´vreux and Ayraud (1982). Slides were directly coloured with Giemsa (4%, pH 6.8) for 8 min. From the about 130 metaphases observed in 30 animals, in which we could confirm the diploid number of 2n ¼ 38, we selected 67 intact and well-spread ones for chromosome analyses and karyotype determination. This was performed using a Nikon Ecclipse E400 microscope with an F-View II digital camera, with Cell ID software associated. Karyotype formula was determined after statistical analysis of chromosome measurements: relative lengths and centromeric indexes. Chromosome nomenclature follows that of Levan et al. (1964). To perform the aneuploidy counts, the remaining animals were divided into 2 groups of nine animals each. The first group was acclimated in water from the natural collection site, and the second group was placed in drinking tap water. Acclimation period was of 2 weeks. The temperature and feeding protocol were the same for both groups. Slides were executed in the same way as referred before and stained with Giemsa. Chromosome number was determined for 100 metaphases and aneuploidy indexes determined for each group, by calculating the percent of cells with abnormal chromosome number in the total pool of cells analyzed.
The diploid number of 2n ¼ 38 for A. cygnea described here (Fig. 2) is coincident with that reported for this species and other Anodonta sp. (A. grandis, A. piscinalis, A. subcircularis, A. woodiana) (Nakamura, 1985; Barsiene, 1994; Woznicki, 2004; Woznicki and Jankun, 2004). The fundamental chromosome arm number (NF) reported in the present paper is similar to that described for four other Unionidae species, and equalled 76 (only bi-armed chromosomes were found) (Nakamura, 1985). The relative length and morphology of chromosomes seems to be similar to A. anatine. In regard to A. woodiana, only the morphology of chromosomes was different (Woznicki, 2004; Woznicki and Jankun, 2004). The main differences between these two karyotypes were the number of submetacentric chromosomes in A. cygnea (12 pairs), which was higher than in A. woodiana (five pairs), and the presence of one pair of subtelocentric chromosomes in A. cygnea. Bivalves are organisms that can accumulate in their tissues several kinds of pollutants. Even in sublethal concentrations, these chemicals may have genotoxic effects that namely translate into aneuploidy (Anderson et al., 1994). These effects are clearly alerting symptoms of environmental adversity (Sokolowski et al., 2004). In our study, the result obtained for the total abnormal metaphase index (aneuploidy and polyploidy) in the lake water was of 19.2% (Fig. 4). In comparison to the reported levels of natural aneuploidy in Anodonta cygnea (Barsiene and Bucinskiene, 2001; Barsiene et al., 2002) we find that these are already above what was expected for an unpolluted site. Thus, we suspect that our collection
Fig. 2. Giemsa-stained diploid metaphase and respective karyotype of Anodonta cygnea.
J. Carrilho et al. / Estuarine, Coastal and Shelf Science 80 (2008) 303–306 Table 1 Chromosome measurements and classification Chromosome pair number
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Relative length
Arm ratio
Centromeric index
Mean
SD
Mean
SD
Mean
SD
6.72 5.98 5.33 4.62 4.15 3.72 7.34 6.41 6.10 5.89 5.51 5.39 5.30 5.08 4.96 4.62 4.31 3.78 4.80
0.88 0.75 0.60 0.55 0.51 0.42 1.03 0.80 0.73 0.71 0.71 0.65 0.61 0.61 0.63 0.53 0.49 0.41 0.58
1.26 1.31 1.26 1.29 1.35 1.21 1.86 2.09 2.07 2.14 2.38 2.10 2.13 2.10 2.21 2.24 2.03 1.91 4.11
0.15 0.21 0.19 0.19 0.18 0.12 0.23 0.25 0.36 0.33 0.31 0.26 0.26 0.44 0.44 0.41 0.28 0.32 1.11
44.48 43.66 44.48 43.95 42.82 45.29 35.13 32.54 33.02 32.14 29.81 32.45 32.17 32.87 32.24 31.38 33.23 35.13 20.32
3.03 3.67 3.61 3.45 3.29 2.40 2.81 2.64 3.68 3.26 2.76 2.56 2.64 5.07 4.42 4.04 2.69 4.17 3.72
Classification
m m m m m m sm sm sm sm sm sm sm sm sm sm sm sm st
305
site was already contaminated, supposedly by the drains of surrounding agricultural fields and residential areas. Nevertheless, the diploid number in the majority of the cells was 38 (similar to that described in other works, as mentioned above). Therefore, we considered the data from this population safe to establish the standard karyotype of this species, based on the centromeric index, the arm ratio and the relative length of the chromosome (Fig. 2 and Table 1). Our studies in Anodonta cygnea, acclimatized in drinking tap water and in natural water from the lake where the individuals were collected, showed significant different indexes of aneuploidy (with special emphasis to hypodiploidy) between individuals in either rearing conditions, with higher values being found in tap water. These results are to be expected if we take in consideration that, drinking tap water, is submitted to a series of treatments before distribution. These include chlorination, chloramination and ozonation. All of these are introducing potential aneugenic compounds in the water (Monarca et al., 2004). Our findings are in agreement with what was described by Scarpato et al. (1990), in this same species, using micronucleus assays. Our study provides, nevertheless, a more refined scenario of the changes in genetic information involved in these processes of
Fig. 3. (a) Hypodiploid metaphase 2n ¼ 35 and respective karyotype. (b) Hyperdiploid metaphase 2n ¼ 42 and respective karyotype. (c) Polyploid metaphase 4n ¼ 76 and respective karyotype.
306
J. Carrilho et al. / Estuarine, Coastal and Shelf Science 80 (2008) 303–306
tap water
a
Acknowledgements
5.9%
The authors acknowledge Prof. Doutora Beatriz Porto for all the assistance and advice, Prof. Doutor Jorge Machado for providing the algal suspension and Sr. Correia for the help in the collection of the animals.
47.1%
References
47% diploid
b
hypodiploid
hyperdiploid
lake water 5.5%
4.1%
9.6%
80.8% diploid
hypodiploid
hyperdiploid
poliploid
Fig. 4. Amount of each type of cell in tap water (a) and in lake water (b).
toxicity. Namely, we can show increases in chromosome number, otherwise undetected by micronuclei testing, and also determine just how much of the chromosome is lost to the micronuclei.
5. Conclusions In conclusion, we can suggest that: (1) aneugenic agents are present in the water from the Lagoa de Mira; (2) there is a higher aneugenic activity in our laboratory tap water, when compared to the lake water; (3) chromosome analysis techniques are a useful tool to study the impact of contaminants referred above making Anodonta cygnea a suitable organism for assessment of cytogenetic damage in aquatic systems. On the other hand, our results also point out to the importance of doing the acclimatizing process of the collected animals in their own natural water to assure the homeostasis of the animal, and consequently avoid systematic bias in future results.
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