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Evidence of non-random chromosome loss in bivalves: Differential chromosomal susceptibility in aneuploid metaphases of Crassostrea angulata (Ostreidae) and Ruditapes decussatus (Veneridae)
Aquaculture 344-349 (2012) 239–241
Contents lists available at SciVerse ScienceDirect
Aquaculture journal homepage: www.elsevier.com/locate/aqua-online
Short communication
Evidence of non-random chromosome loss in bivalves: Differential chromosomal susceptibility in aneuploid metaphases of Crassostrea angulata (Ostreidae) and Ruditapes decussatus (Veneridae) Joana Teixeira de Sousa a, Sandra Joaquim a, Domitília Matias a, Radhouan Ben-Hamadou b, Alexandra Leitão a,⁎ a b
Instituto Nacional de Recursos Biológicos (INRB, I.P.) /L-IPIMAR, Av. 5 de Outubro s/n, P-8700-305 Olhão, Portugal Centro de Ciências do Mar (CCMAR), EcoReach Research Group, Universidade do Algarve, Campus de Gambelas, P-8005-139 Faro, Portugal
a r t i c l e
i n f o
Article history: Received 30 September 2011 Received in revised form 16 March 2012 Accepted 26 March 2012 Available online 3 April 2012 Keywords: Bivalve Aneuploidy Growth Chromosome In situ restriction enzyme banding
a b s t r a c t Aneuploidy is a cytogenetic phenomenon known as an abnormal diploid chromosome number. A negative relationship between growth rate, one of the biggest problems faced by bivalve producers, and this phenomenon was already verified for two oyster species, the Japanese oyster Crassostrea gigas, the Portuguese oyster Crassostrea angulata and their interspecific hybrids and, more recently, in the carpet shell clam Ruditapes decussatus. The main objective of this study was to assess whether chromosome losses in aneuploid situations could be explained by differential chromosomal susceptibility, as previously reported in the oyster C. gigas. Thereby, we applied the restriction enzyme (RE) digestion chromosome banding technique to aneuploid karyotypes of R. decussatus and C. angulata, in order to identify the missing chromosomes. The results of this study showed that 4 out of the 19 chromosome pairs (viz. 1, 6, 12, and 19) of R. decussatus and 3 out of the 10 chromosome pairs (viz. 1, 9, and 10) of C. angulata were preferentially affected by the loss of one homologous chromosome. The chromosomal loss in C. angulata was very similar to the one previously observed in C. gigas. These results open a new field for further research in order to have a better understanding of the aneuploidy phenomenon in bivalves and particularly its negative relationship with growth rate. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Aneuploidy is a cytogenetic phenomenon known as an abnormal diploid chromosome number that involves the loss (hypoploidy) or gain (hyperploidy) of one or more individual chromosomes. A negative relationship between growth rate and this phenomenon (hypoploidy) was already put in evidence for two commercially important oyster species: the Japanese oyster Crassostrea gigas (e.g. Leitão et al., 2001a; Thiriot-Quiévreux et al., 1988; Zouros et al., 1996) the Portuguese oyster C. angulata and their interspecific hybrids (Batista et al., 2007). Moreover, the hypothesis of a genetic basis for the level of aneuploidy was also suggested for those species (Batista et al., 2007; Leitão et al., 2001b). More recently, a negative correlation with growth rate was also put in evidence in the carpet shell clam Ruditapes decussatus, one of the most important bivalve species in Portugal and other Southern European countries (Teixeira de Sousa et al., 2011). The study of this phenomenon is of particular importance, since the variability of growth rate is one of the biggest problems faced by bivalve producers.
⁎ Corresponding author. Tel.: + 351 281 326951; fax: + 351 281 324028. E-mail address: [email protected] (A. Leitão). 0044-8486/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2012.03.031
In order to try to clarify the nature of the aneuploidy phenomenon, previous studies performed in C. gigas showed that only 4 of the 10 chromosome pairs were affected by chromosome loss (viz. 1, 5, 9, and 10), indicating that chromosomal loss in that species was not random, but reflected differential chromosome susceptibility (Leitão et al., 2001c). The main objective of this study was then to assess whether this non-random chromosome loss was also evident in R. decussatus and C. angulata, by applying the molecular cytogenetic technique of digestion banding with restriction enzymes, already successfully applied in bivalves, mainly in oysters (Bouilly et al., 2005; Cross et al., 2005; Leitão et al., 2004) and veneroids (Leitão et al., 2006), for the identification of the missing chromosomes in aneuploid karyotypes of these two species. 2. Materials and methods Juvenile specimens of R. decussatus resulting from a single spawning of wild parents, with 10 females and 8 males in a total of 30 individuals stimulated, induced in the experimental bivalve hatchery of National Institute of Biological Resources (INRB, I.P./L-IPIMAR) in Tavira (Portugal) and juvenile specimens of C. angulata from Rio Sado, Portugal, were incubated for 8–10 h in seawater containing 0.005%
240
J.T. de Sousa et al. / Aquaculture 344-349 (2012) 239–241
colchicine. Chromosome preparations were obtained following the air drying technique of Thiriot-Quiévreux and Ayraud (1982). Chromosome counts were made directly by microscope observation (Nikon Eclipse 80i with camera image acquisition incorporated Nikon DS-Fi1) on a large number of apparently intact metaphases, in order to avoid any bias of chromosomal loss due to artifacts of the technique used. The aneuploidy level determined corresponds to the percentage of aneuploid cells in a total number of cells analyzed in the gill tissue. For the RE digestion chromosome banding technique, 28 individuals of R. decussatus and 15 individuals of C. angulata were studied, with 32 and 44 aneuploid metaphases analyzed, respectively. Slides were treated with the RE HaeIII (GG/CC) following Leitão et al. (2004). Control slides were subjected to the same treatment as described above but incubated only with buffer. Karyotypes of aneuploid metaphases were performed taking into consideration standard measurements of chromosome pairs (measurements of size and centromeric index) and RE digestion chromosome banding pattern. We applied the McNemar test to a contingency table built with the aneuploid metaphases distributed according to the different combinations of loss of specific chromosomes. 3. Results and discussion R. decussatus has a diploid number of 38 and C. angulata a diploid number of 20 chromosomes; however, in previous studies hypoploid cells were observed in both species. The frequency of hypodiploid cells determined in those studies was in average 38.5% for R. decussatus (Teixeira de Sousa et al., 2011) and 20% for C. angulata (Batista et al., 2007). The in situ RE banding procedure, in this study, revealed clear and characteristic bands, allowing the unambiguous individual identification of all the chromosome pairs. Karyotypes of RE banded aneuploid metaphases with 2n = 34, 35, 36 and 37 chromosomes for R. decussatus and 2n = 17, 18 and 19 chromosomes for C. angulata were performed. Chromosome loss from either one pair or more than one pair per karyotype was observed; however, the loss of both homologues of one chromosomal pair was not observed in any of the karyotypes of both species. For R. decussatus, the analysis of the aneuploid karyotypes showed that mainly 4 out of the 19 chromosome pairs were affected by chromosome loss (pairs 1, 6, 12 and 19), with the highest percentage of chromosome loss being found in the smallest subtelocentric (47%) chromosomal pair (Tables 1 and 2). For C. angulata, mainly 3 out of the 10 chromosome pairs were affected by chromosome loss (pairs 1, 9 and 10), with the highest percentage of chromosome loss being found in the largest (44%) chromosomal pair (Tables 1 and 2). The McNemar test revealed (α = 0.005) that, the chromosome losses of pairs 1, 6, 12 and 19 in R. decussatus and 1, 9 and 10 in C. angulata were not random. Moreover the results highlighted that the probability of losing chromosome 19 in R. decussatus (47%) is significantly higher than the probability of losing chromosome 6 (27%) in this species. The same was observed for chromosome 1 (44%) in C. angulata with a significantly higher probability of loss than chromosome 10 (26%). The results for C. angulata were very similar to the ones observed in C. gigas (Leitão et al., 2001c), where the study of 95 G-banded aneuploid karyotypes also showed that pairs 1, 9 and 10, which were lost in 56, 33 and 44% of cases, respectively, were the ones more affected by the loss of one homologous chromosome. These results were not unexpected, knowing the high taxonomical proximity of these two oyster species (e.g. Boudry et al., 1998; Huvet et al., 2004).
Table 2 Percentage of chromosome loss in Ruditapes decussatus and Crassostrea angulata, for each lost pair in RE banded aneuploid karyotypes. Species
Pairs
Percentage of chromosome loss
No. of RE banded karyotypes
R. decussatus
1 6 12 19 1 9 10
37% 27% 40% 47% 44% 33% 26%
32
C. angulata
44
The evidence of a non-random chromosome loss in these three commercially important bivalve species, C. gigas, C. angulata and R. decussatus, emphasized by this and previous studies opens a new field of research for this phenomenon and particularly its negative relationship with growth rate. Such a non-random chromosome loss in aneuploid situations had already been observed in humans and plants (Cheng and Murata, 2002; Sun et al., 2006; Takeuchi et al., 2009). Indeed, certain chromosome pairs were lost more often than expected under the assumption of non randomness of segregation. Several hypotheses can be formulated to explain the non-random chromosome loss observed, such as: (i) small chromosome size (for pairs 6 and 19 in R. decussatus and pair 10 in C. angulata). Indeed, a negative correlation between chromosome size and chromosome loss was previously observed in humans, since the smaller chromosomes are more likely to be achiasmate than larger ones (Sun et al., 2006); (ii) largely heterochromatic chromosomes, which mainly could justify the loss of the largest chromosomal pair (pair 1 in both species) and yet the survival of the cell. Indeed, heterochromatin represents DNA that is permanently silenced, which consists primarily of repeated sequences and contains relatively few genes (Karp, 2010); (iii) presence of NORs (nucleolus organizer region) and non disjunction. A relationship between NOR association and the non disjunction phenomenon, a cause of aneuploidy, has been suggested by several authors (e.g., Verma, 1990; Yashwanth et al., 2010). Indeed the involvement of NOR-bearing chromosomes in somatic associations has been observed in several species. The main function of those associations seems to be the cooperation between two or more NORs in the formation of same nucleolus (e.g., Tatewaki et al., 2002). In C. angulata (Leitão et al., 1999) and also in C. gigas (Thiriot-Quiévreux and Insua, 1992) the NORs are located on pair 10 which could explain the high level of chromosome loss observed for this pair. Given the high rate of chromosome loss observed in several pairs, we can suggest an imbalance in gene dosage caused by aneuploidy. Indeed, the massive alteration of gene expression has already showed to be the cause of qualitative changes in physiology and metabolism of cells and tissues (Rasnick and Duesberg, 1999). Recent studies have showed that phenotypic changes occur due to quantitative changes in the gene expression pattern, caused by aneuploidy (Makarevich and Harris, 2010). Further studies are presently being conducted by our team in order to clarify the nature of the non-random chromosomal loss observed, such as the physical mapping of genes with specific traits related to growth and the location of repetitive DNA sequences, by fluorescence in situ hybridization (FISH). Although the heritability of this phenomenon on these species should be prior investigated, the evidence of the genetic basis demonstrated previously for C. angulata and C. gigas
Table 1 Combinations of chromosome loss in Ruditapes decussatus and Crassostrea angulata aneuploid karyotypes. Combinations of chromosome loss R. decussatus C. angulata
1 1
6 9
12 10
15 1/10
19 9/10
1/12 1/5/10
1/19
6/19
12/19
6/19
9/19
9/12
7/19
1/10/12
1/11/19
5/7/17
6/9/12/19
1/6/7/9
6/7/13/15
1/6/12/19
J.T. de Sousa et al. / Aquaculture 344-349 (2012) 239–241
(e.g., Batista et al., 2007), and the non-random chromosome loss and negative relationship between aneuploidy and growth rate, lead us to suggest that aneuploidy should be one of the parameters to be taken into account in the design of future breeding programs of these commercially important bivalve species, mainly by the selection of broodstock with low levels of aneuploidy. Acknowledgments This work was supported by the projects PTDC/MAR/72163/2006: FCOMP-01-0124-FEDER-007384 and “Ciência 2007 program.” We are grateful to Margarete Matias and João Maurício Teixeira for the excellent technical assistance. The authors are also grateful to A. Good for revising the English. Thanks are also due to the three anonymous referees for valuable comments that greatly improved the manuscript. References Batista, F., Leitão, A., Fonseca, V., Ben-Hamadou, R., Ruano, F., Henriques, M., GuedesPinto, H., Boudry, P., 2007. Individual relationship between aneuploidy of gill cells and growth rate in cupped oysters Crassostrea angulata, C. gigas and their reciprocal hybrids. Journal of Experimental Marine Biology and Ecology 352, 226–233. Boudry, P., Heurtebise, S., Collet, B., Cornette, F., Gerard, A., 1998. Differentiation between populations of the Portuguese oyster, Crassostrea angulata (Lamark) and the Pacific oyster, Crassostrea gigas (Thunberg), revealed by mtDNA RFLP analysis. Journal of Experimental Marine Biology and Ecology 226, 279–291. Bouilly, K., Leitão, A., Chaves, R., Guedes-Pinto, H., Boudry, P., Lapègue, S., 2005. Endonuclease banding reveals that atrazine induced aneuploidy resembles spontaneous chromosome loss in Crassostrea gigas. Genome 48, 177–180. Cheng, Z.J., Murata, M., 2002. Loss of chromosomes 2R and 5RS in octoploid triticale selected for agronomic traits. Genes & Genetic System 77, 23–29. Cross, I., Sánchez, I., Rebordinos, L., 2005. Molecular and cytogenetic characterization of Crassostrea angulata chromosomes. Aquaculture 247, 135–144. Huvet, A., Fabioux, C., McCombie, H., Lapègue, S., Boudry, P., 2004. Natural hybridization in genetically differentiated populations of Crassostrea gigas and C. angulata highlighted by sequence variation in flanking regions of a microsatellite locus. Marine Ecology Progress Series 272, 141–152. Karp, G., 2010. Cell Biology, sixth ed. John Wiley & Sons (Asia) Pte Ltd. Leitão, A., Boudry, P., Labal, J.P., Thiriot-Quiévreux, C., 1999. Comparative karyological study of cupped oyster species. Malacologia 41, 175–186.
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Leitão, A., Boudry, P., Thiriot-Quiévreux, C., 2001a. Negative correlation between aneuploidy and growth in the Pacific oyster Crassostrea gigas: ten years of evidence. Aquaculture 193, 39–48. Leitão, A., Boudry, P., McCombie, H., Gérard, A., Thiriot-Quiévreux, C., 2001b. Experimental evidence for a genetic basis to differences in aneuploidy in the Pacific Oyster (Crassostrea gigas). Aquatic Living Resources 14, 233–237. Leitão, A., Boudry, P., Thiriot-Quiévreux, C., 2001c. Evidence of differential chromosome loss in aneuploid karyotypes of the Pacific oyster Crassostrea gigas. Genome 44, 735–737. Leitão, A., Chaves, R., Santos, S., Guedes-Pinto, H., Boudry, P., 2004. Restriction enzyme digestion chromosome banding in Crassostrea and Ostrea species: comparative karyological analysis within Ostreidae. Genome 47, 781–788. Leitão, A., Chaves, R., Matias, D., Joaquim, S., Ruano, F., Guedes-Pinto, H., 2006. Restriction enzyme digestion chromosome banding on two commercially important venerid bivalve species: Ruditapes decussatus and Cerastoderma edule. Journal of Shellfish Research 3, 857–863. Makarevich, I., Harris, C., 2010. Aneuploidy tissue specific qualitative changes in global gene expression patterns in maize. Plant Physiology 109. Rasnick, D., Duesberg, P.H., 1999. How aneuploidy affects metabolic control and causes cancer. Biochemical Journal 340, 621–630. Sun, F., Oliver-Bonet, M., Liehr, T., Starke, H., Turek, P., Ko, E., Rademaker, A., Martin, R.H., 2006. Analysis of non-crossover bivalents in pachytene cells from 10 normal men. Human Reproduction 21 (9), 2335–2339. Takeuchi, M., Takeuchi, K., Ozawa, Y., Kohara, A., Mizusawa, H., 2009. Aneuploidy in immortalized human mesenchymal stem cells with non-random loss of chromosome 13 in culture. In Vitro Cellular Developmental Biology. Animal 45, 290–299. Tatewaki, R., Kagohashi, Y., Furuse, K., Otani, H., 2002. Chromosome analysis of blastocysts cultured under the diabetic condition. Congenital Anomalies 42, 21–26. Teixeira de Sousa, J., Matias, D., Joaquim, S., Ben-Hamadou, R., Leitão, A., 2011. Growth variation in bivalves: new insights into growth, physiology and somatic aneuploidy in the carpet shell Ruditapes decussatus. Journal of Experimental Marine Biology and Ecology 406, 46–53. Thiriot-Quiévreux, C., Ayraud, N., 1982. Les caryotypes de quelques espèces de Bivalves et de Gastéropodes marins. Marine Biology 70, 165–172. Thiriot-Quiévreux, C., Insua, A., 1992. Nucleolar organiser region variation in the chromosomes of three oyster species. Journal of Experimental Marine Biology and Ecology 157, 33–40. Thiriot-Quiévreux, C., Noel, T., Bougrier, S., Dallot, S., 1988. Relationships between aneuploidy anf growth rate in pair matings of the oyster Crassostrea gigas. Aquaculture 75, 89–96. Verma, R., 1990. The Genome. VCH Publishers incorporated. Yashwanth, R., Chandra, N., Gopinath, P.M., 2010. Satellite associations in Down syndrome. International Journal of Human Genetics 10 (1–3), 101–104. Zouros, E., Thiriot-Quievreux, C., Kotoulas, G., 1996. The negative correlation between somatic aneuploidy and growth in the oyster Crassostrea gigas and implications for the effects of induced polyploidization. Genetics Research 68, 109–116.
Contents lists available at SciVerse ScienceDirect
Aquaculture journal homepage: www.elsevier.com/locate/aqua-online
Short communication
Evidence of non-random chromosome loss in bivalves: Differential chromosomal susceptibility in aneuploid metaphases of Crassostrea angulata (Ostreidae) and Ruditapes decussatus (Veneridae) Joana Teixeira de Sousa a, Sandra Joaquim a, Domitília Matias a, Radhouan Ben-Hamadou b, Alexandra Leitão a,⁎ a b
Instituto Nacional de Recursos Biológicos (INRB, I.P.) /L-IPIMAR, Av. 5 de Outubro s/n, P-8700-305 Olhão, Portugal Centro de Ciências do Mar (CCMAR), EcoReach Research Group, Universidade do Algarve, Campus de Gambelas, P-8005-139 Faro, Portugal
a r t i c l e
i n f o
Article history: Received 30 September 2011 Received in revised form 16 March 2012 Accepted 26 March 2012 Available online 3 April 2012 Keywords: Bivalve Aneuploidy Growth Chromosome In situ restriction enzyme banding
a b s t r a c t Aneuploidy is a cytogenetic phenomenon known as an abnormal diploid chromosome number. A negative relationship between growth rate, one of the biggest problems faced by bivalve producers, and this phenomenon was already verified for two oyster species, the Japanese oyster Crassostrea gigas, the Portuguese oyster Crassostrea angulata and their interspecific hybrids and, more recently, in the carpet shell clam Ruditapes decussatus. The main objective of this study was to assess whether chromosome losses in aneuploid situations could be explained by differential chromosomal susceptibility, as previously reported in the oyster C. gigas. Thereby, we applied the restriction enzyme (RE) digestion chromosome banding technique to aneuploid karyotypes of R. decussatus and C. angulata, in order to identify the missing chromosomes. The results of this study showed that 4 out of the 19 chromosome pairs (viz. 1, 6, 12, and 19) of R. decussatus and 3 out of the 10 chromosome pairs (viz. 1, 9, and 10) of C. angulata were preferentially affected by the loss of one homologous chromosome. The chromosomal loss in C. angulata was very similar to the one previously observed in C. gigas. These results open a new field for further research in order to have a better understanding of the aneuploidy phenomenon in bivalves and particularly its negative relationship with growth rate. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Aneuploidy is a cytogenetic phenomenon known as an abnormal diploid chromosome number that involves the loss (hypoploidy) or gain (hyperploidy) of one or more individual chromosomes. A negative relationship between growth rate and this phenomenon (hypoploidy) was already put in evidence for two commercially important oyster species: the Japanese oyster Crassostrea gigas (e.g. Leitão et al., 2001a; Thiriot-Quiévreux et al., 1988; Zouros et al., 1996) the Portuguese oyster C. angulata and their interspecific hybrids (Batista et al., 2007). Moreover, the hypothesis of a genetic basis for the level of aneuploidy was also suggested for those species (Batista et al., 2007; Leitão et al., 2001b). More recently, a negative correlation with growth rate was also put in evidence in the carpet shell clam Ruditapes decussatus, one of the most important bivalve species in Portugal and other Southern European countries (Teixeira de Sousa et al., 2011). The study of this phenomenon is of particular importance, since the variability of growth rate is one of the biggest problems faced by bivalve producers.
⁎ Corresponding author. Tel.: + 351 281 326951; fax: + 351 281 324028. E-mail address: [email protected] (A. Leitão). 0044-8486/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2012.03.031
In order to try to clarify the nature of the aneuploidy phenomenon, previous studies performed in C. gigas showed that only 4 of the 10 chromosome pairs were affected by chromosome loss (viz. 1, 5, 9, and 10), indicating that chromosomal loss in that species was not random, but reflected differential chromosome susceptibility (Leitão et al., 2001c). The main objective of this study was then to assess whether this non-random chromosome loss was also evident in R. decussatus and C. angulata, by applying the molecular cytogenetic technique of digestion banding with restriction enzymes, already successfully applied in bivalves, mainly in oysters (Bouilly et al., 2005; Cross et al., 2005; Leitão et al., 2004) and veneroids (Leitão et al., 2006), for the identification of the missing chromosomes in aneuploid karyotypes of these two species. 2. Materials and methods Juvenile specimens of R. decussatus resulting from a single spawning of wild parents, with 10 females and 8 males in a total of 30 individuals stimulated, induced in the experimental bivalve hatchery of National Institute of Biological Resources (INRB, I.P./L-IPIMAR) in Tavira (Portugal) and juvenile specimens of C. angulata from Rio Sado, Portugal, were incubated for 8–10 h in seawater containing 0.005%
240
J.T. de Sousa et al. / Aquaculture 344-349 (2012) 239–241
colchicine. Chromosome preparations were obtained following the air drying technique of Thiriot-Quiévreux and Ayraud (1982). Chromosome counts were made directly by microscope observation (Nikon Eclipse 80i with camera image acquisition incorporated Nikon DS-Fi1) on a large number of apparently intact metaphases, in order to avoid any bias of chromosomal loss due to artifacts of the technique used. The aneuploidy level determined corresponds to the percentage of aneuploid cells in a total number of cells analyzed in the gill tissue. For the RE digestion chromosome banding technique, 28 individuals of R. decussatus and 15 individuals of C. angulata were studied, with 32 and 44 aneuploid metaphases analyzed, respectively. Slides were treated with the RE HaeIII (GG/CC) following Leitão et al. (2004). Control slides were subjected to the same treatment as described above but incubated only with buffer. Karyotypes of aneuploid metaphases were performed taking into consideration standard measurements of chromosome pairs (measurements of size and centromeric index) and RE digestion chromosome banding pattern. We applied the McNemar test to a contingency table built with the aneuploid metaphases distributed according to the different combinations of loss of specific chromosomes. 3. Results and discussion R. decussatus has a diploid number of 38 and C. angulata a diploid number of 20 chromosomes; however, in previous studies hypoploid cells were observed in both species. The frequency of hypodiploid cells determined in those studies was in average 38.5% for R. decussatus (Teixeira de Sousa et al., 2011) and 20% for C. angulata (Batista et al., 2007). The in situ RE banding procedure, in this study, revealed clear and characteristic bands, allowing the unambiguous individual identification of all the chromosome pairs. Karyotypes of RE banded aneuploid metaphases with 2n = 34, 35, 36 and 37 chromosomes for R. decussatus and 2n = 17, 18 and 19 chromosomes for C. angulata were performed. Chromosome loss from either one pair or more than one pair per karyotype was observed; however, the loss of both homologues of one chromosomal pair was not observed in any of the karyotypes of both species. For R. decussatus, the analysis of the aneuploid karyotypes showed that mainly 4 out of the 19 chromosome pairs were affected by chromosome loss (pairs 1, 6, 12 and 19), with the highest percentage of chromosome loss being found in the smallest subtelocentric (47%) chromosomal pair (Tables 1 and 2). For C. angulata, mainly 3 out of the 10 chromosome pairs were affected by chromosome loss (pairs 1, 9 and 10), with the highest percentage of chromosome loss being found in the largest (44%) chromosomal pair (Tables 1 and 2). The McNemar test revealed (α = 0.005) that, the chromosome losses of pairs 1, 6, 12 and 19 in R. decussatus and 1, 9 and 10 in C. angulata were not random. Moreover the results highlighted that the probability of losing chromosome 19 in R. decussatus (47%) is significantly higher than the probability of losing chromosome 6 (27%) in this species. The same was observed for chromosome 1 (44%) in C. angulata with a significantly higher probability of loss than chromosome 10 (26%). The results for C. angulata were very similar to the ones observed in C. gigas (Leitão et al., 2001c), where the study of 95 G-banded aneuploid karyotypes also showed that pairs 1, 9 and 10, which were lost in 56, 33 and 44% of cases, respectively, were the ones more affected by the loss of one homologous chromosome. These results were not unexpected, knowing the high taxonomical proximity of these two oyster species (e.g. Boudry et al., 1998; Huvet et al., 2004).
Table 2 Percentage of chromosome loss in Ruditapes decussatus and Crassostrea angulata, for each lost pair in RE banded aneuploid karyotypes. Species
Pairs
Percentage of chromosome loss
No. of RE banded karyotypes
R. decussatus
1 6 12 19 1 9 10
37% 27% 40% 47% 44% 33% 26%
32
C. angulata
44
The evidence of a non-random chromosome loss in these three commercially important bivalve species, C. gigas, C. angulata and R. decussatus, emphasized by this and previous studies opens a new field of research for this phenomenon and particularly its negative relationship with growth rate. Such a non-random chromosome loss in aneuploid situations had already been observed in humans and plants (Cheng and Murata, 2002; Sun et al., 2006; Takeuchi et al., 2009). Indeed, certain chromosome pairs were lost more often than expected under the assumption of non randomness of segregation. Several hypotheses can be formulated to explain the non-random chromosome loss observed, such as: (i) small chromosome size (for pairs 6 and 19 in R. decussatus and pair 10 in C. angulata). Indeed, a negative correlation between chromosome size and chromosome loss was previously observed in humans, since the smaller chromosomes are more likely to be achiasmate than larger ones (Sun et al., 2006); (ii) largely heterochromatic chromosomes, which mainly could justify the loss of the largest chromosomal pair (pair 1 in both species) and yet the survival of the cell. Indeed, heterochromatin represents DNA that is permanently silenced, which consists primarily of repeated sequences and contains relatively few genes (Karp, 2010); (iii) presence of NORs (nucleolus organizer region) and non disjunction. A relationship between NOR association and the non disjunction phenomenon, a cause of aneuploidy, has been suggested by several authors (e.g., Verma, 1990; Yashwanth et al., 2010). Indeed the involvement of NOR-bearing chromosomes in somatic associations has been observed in several species. The main function of those associations seems to be the cooperation between two or more NORs in the formation of same nucleolus (e.g., Tatewaki et al., 2002). In C. angulata (Leitão et al., 1999) and also in C. gigas (Thiriot-Quiévreux and Insua, 1992) the NORs are located on pair 10 which could explain the high level of chromosome loss observed for this pair. Given the high rate of chromosome loss observed in several pairs, we can suggest an imbalance in gene dosage caused by aneuploidy. Indeed, the massive alteration of gene expression has already showed to be the cause of qualitative changes in physiology and metabolism of cells and tissues (Rasnick and Duesberg, 1999). Recent studies have showed that phenotypic changes occur due to quantitative changes in the gene expression pattern, caused by aneuploidy (Makarevich and Harris, 2010). Further studies are presently being conducted by our team in order to clarify the nature of the non-random chromosomal loss observed, such as the physical mapping of genes with specific traits related to growth and the location of repetitive DNA sequences, by fluorescence in situ hybridization (FISH). Although the heritability of this phenomenon on these species should be prior investigated, the evidence of the genetic basis demonstrated previously for C. angulata and C. gigas
Table 1 Combinations of chromosome loss in Ruditapes decussatus and Crassostrea angulata aneuploid karyotypes. Combinations of chromosome loss R. decussatus C. angulata
1 1
6 9
12 10
15 1/10
19 9/10
1/12 1/5/10
1/19
6/19
12/19
6/19
9/19
9/12
7/19
1/10/12
1/11/19
5/7/17
6/9/12/19
1/6/7/9
6/7/13/15
1/6/12/19
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(e.g., Batista et al., 2007), and the non-random chromosome loss and negative relationship between aneuploidy and growth rate, lead us to suggest that aneuploidy should be one of the parameters to be taken into account in the design of future breeding programs of these commercially important bivalve species, mainly by the selection of broodstock with low levels of aneuploidy. Acknowledgments This work was supported by the projects PTDC/MAR/72163/2006: FCOMP-01-0124-FEDER-007384 and “Ciência 2007 program.” We are grateful to Margarete Matias and João Maurício Teixeira for the excellent technical assistance. The authors are also grateful to A. Good for revising the English. Thanks are also due to the three anonymous referees for valuable comments that greatly improved the manuscript. References Batista, F., Leitão, A., Fonseca, V., Ben-Hamadou, R., Ruano, F., Henriques, M., GuedesPinto, H., Boudry, P., 2007. Individual relationship between aneuploidy of gill cells and growth rate in cupped oysters Crassostrea angulata, C. gigas and their reciprocal hybrids. Journal of Experimental Marine Biology and Ecology 352, 226–233. Boudry, P., Heurtebise, S., Collet, B., Cornette, F., Gerard, A., 1998. Differentiation between populations of the Portuguese oyster, Crassostrea angulata (Lamark) and the Pacific oyster, Crassostrea gigas (Thunberg), revealed by mtDNA RFLP analysis. Journal of Experimental Marine Biology and Ecology 226, 279–291. Bouilly, K., Leitão, A., Chaves, R., Guedes-Pinto, H., Boudry, P., Lapègue, S., 2005. Endonuclease banding reveals that atrazine induced aneuploidy resembles spontaneous chromosome loss in Crassostrea gigas. Genome 48, 177–180. Cheng, Z.J., Murata, M., 2002. Loss of chromosomes 2R and 5RS in octoploid triticale selected for agronomic traits. Genes & Genetic System 77, 23–29. Cross, I., Sánchez, I., Rebordinos, L., 2005. Molecular and cytogenetic characterization of Crassostrea angulata chromosomes. Aquaculture 247, 135–144. Huvet, A., Fabioux, C., McCombie, H., Lapègue, S., Boudry, P., 2004. Natural hybridization in genetically differentiated populations of Crassostrea gigas and C. angulata highlighted by sequence variation in flanking regions of a microsatellite locus. Marine Ecology Progress Series 272, 141–152. Karp, G., 2010. Cell Biology, sixth ed. John Wiley & Sons (Asia) Pte Ltd. Leitão, A., Boudry, P., Labal, J.P., Thiriot-Quiévreux, C., 1999. Comparative karyological study of cupped oyster species. Malacologia 41, 175–186.
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