Chromosome Research 1997, 5, 268–273 Analysis of NORs and NOR-associated heterochromatin in the mussel Mytilus galloprovincialis Lmk M. J. Martı ´nez-Expo ´sito, J. Me ´ndez & J. J. Pasantes Received 21 October 1996; received in revised form and accepted for publication by M. Schmid 18 February 1997 The chromosomes of the mussel Mytilus galloprovin- cialis were analysed by means of chromomycin A3 (CMA), distamycin A/DAPI (DA/DAPI), DAPI/actinomy- cin D (DAPI/AMD) and chromomycin A3/distamycin A/DAPI (CDD) fluorescence banding techniques, C- banding, silver staining, N-banding and in situ hybri- dization with 18S 28S rDNA and telomere probes. 18S 28S rDNA clusters were located on the telomeres of two pairs of submeta/subtelocentric chromosomes. The nucleolar organizing regions (NORs) were asso- ciated with bright CMA fluorescence, dull DAPI fluor- escence and C- and N-positive bands, but not all four NOR-associated heterochromatin bands showed bright CMA fluorescence in a given cell; intra- and interindividual variability was found in this character. Additional non-ribosomal C-bands did not show any differential fluorescent behaviour. Key words: fluorochrome staining, heterochromatin, in situ hybridization, Mytilus galloprovincialis, nucleolar organizing regions Introduction Although banding techniques have proved to be very useful in the study of chromosomes, most cytogenetic data in bivalve molluscs have been obtained by con- ventional staining procedures. This is primarily be- cause of the general difficulties in working with the chromosomes of these animals. The few cytogenetic studies performed to date on the different chromatin classes in the karyotypes of bivalve molluscs have made use of Ag-NOR staining and/or C-banding (Dixon et al. 1986, Dixon & McFadzen 1987, Jacobi et al. 1990, Insua & Thiriot-Quie ´vreux 1991, Thiriot- Quie ´vreux & Insua 1992, Martı ´nez-Expo ´sito et al. 1994, Pasantes et al. 1996); only Martı ´nez-Lage et al. (1994, 1995) used fluorochrome staining. In previous studies we have detected variable NOR activity (Martı ´nez-Expo ´sito et al. 1994) and C-band polymorphisms (Pasantes et al. 1996) in the chromo- somes of the mussel Mytilus galloprovincialis. To ana- lyse the structure and variability of these bands, we have applied C- and N-banding, and silver- and fluoro- chrome-staining techniques to fixed mitotic chromo- somes and interphase nuclei of this species. We also used in situ hybridization with a 18S 28S rDNA probe to confirm the localization of these sequences. Materials and methods Individuals of a species of mussel, Mytilus galloprovincialis (2n 28), were collected from an intertidal population in Balcobo (Galicia, NW Spain). A total of 50 animals (1–2 cm shell length) were maintained for 12 days in the laboratory in 5l tanks of aerated, filtered sea water at 20 1 C, and they were fed on a suspension of mixed algal cells (Tetraselmis suecica, Isochrysis galbana, and Phaeodactylum tricornutum) to promote somatic growth. After a colchicine (0.005%) treatment for 10 h, mussels were briefly rinsed in clean sea water and the gills were excised. The tissue was then immersed in diluted sea water for 1 h and fixed in Carnoy’s fixative for 1 h. Chromosome spreads were obtained by dissociating gill tissue in 45% acetic acid, pipet- ting suspension drops onto slides heated to 43 C and air- drying. For fluorescent pattern analysis, three different combina- tions of fluorochromes and specific counterstains were used. In some slides, chromosomes were stained by the chromo- mycin A3/distamycin A/DAPI (CDD) counterstaining meth- od (Schweizer 1980). After staining, the slides were aged in the dark for 4–6 days before examination. By photographing suitable metaphase spreads using two different filter combi- nations, both the CMA and the distamycin A/DAPI (DA/ DAPI) staining behaviour were sequentially recorded. Other slides were stained with CMA, aged for 4–6 days and photo- graphed (Schweizer 1976). After destaining, slides were stained with DAPI, aged for 2 days and photographed again. Whatever the method used and subsequent to the photogra- phy, the preparations were destained and the same meta- phases were further analysed by the DAPI/actinomycin D (DAPI/AMD) staining method (Schweizer 1976). Slide visua- lization and photography were performed using a Nikon Michrophot FXA equipped with an epifluorescence system. Some of the slides previously examined by fluorescent microscopy were C-banded following the technique descri- M. J. Martı ´nez-Expo ´sito and J. J. Pasantes (corresponding author) are at the Dpto. Bioloxı ´a Fundamental, Xene ´tica, Universidade de Vigo, E-36200 Vigo, Spain. Tel: ( 34) 86 812577; Fax: ( 34) 86 812556; Email: pasantes@setei.uvigo.es. J. Me ´ndez is at the Dpto. Bioloxı ´a Celular e Molecular, Xene ´tica, Universidade da Corun ˜a, E-15071 A Corun ˜a, Spain. 268 Chromosome Research Vol 5 1997