Band View/FISH View software

At least 15–20 metaphase spreads from each individual were studied using a Nikon Eclipse 90i fluorescence microscope equipped with ProgRes MFcool camera (Jenoptic, Jena, Germany) and a Zeiss Axio Imager.A1 fluorescence microscope equipped with a fluorescent lamp and a digital camera (Applied Spectral Imaging, Galilee, Israel). Both microscopes were supported by appropriate filter set for the multicolor FISH technique. A Lucia software ver. 2.0 (Laboratory Imaging, Prague, Czech Republic) and Band View/FISH View software (Applied Spectral Imaging, Galilee, Israel) were also used for the capturing and the electronic processing of the images. Post-processing elaboration of all the figures were made using CorelDRAW^® Graphics Suite 11 (Corel Corporation, Ottawa, Canada). All the chromosomes were classified according to Levan et al. (1964 (link)), where metacentric (m) and submetacentric (sm) chromosomes were considered as bi-armed, while subtelocentric (st) and acrocentric (a) chromosomes as uni-armed. A total of 423 chromosome metaphase spreads were investigated in the study.
Voucher specimens have been preserved frozen at the Department of Zoology and Department of Ichthyology, University of Warmia and Mazury in Olsztyn, Poland.

We randomly sampled ten larvae from each treatment and control group and placed them in a 0.025% solution of colchicine for 4 h at room temperature (16 °C). Larvae were alive in the colchicine solution. The heads of larvae were dissected and hypotonized in 0.025% KCl for 45 min (in the refrigerator). Then, they were fixed in methanol/acetic acid 3:1 solution three times (in the refrigerator): the first time for 30 min and then two times for 15 min. The fixed heads were homogenized in Eppendorf tubes and the cell suspensions were placed in a microscope slide. Dried chromosome preparations were stained with 5% Giemsa solution for 20 min. We analysed at least ten good-quality metaphase spreads from each cytogenetically studied larva under a Zeiss Axio Imager A1 microscope equipped with a fluorescent lamp and a digital camera; captured images were processed electronically using Band View/FISH View software (Applied Spectral Imaging, Carlsbad, CA, USA). The number of chromosomes was counted in all treatment and control groups for ploidy estimation.

Metaphase plates were examined using Nikon Optiphot, Nikon 90i (Nikon, Japan) and Zeiss Axio Imager.A1 (Zeiss, Germany) microscopes equipped with epi-fluorescence and the digital cameras. Pictures were acquired using a monochromatic ProgRes MFcool camera (Jenoptic, Germany) controlled by a Lucia software (Laboratory Imaging, Czech Republic). Images of chromosomes after PNA-FISH and PRINS were captured and the electronic processing of the images was performed using Band View/FISH View software (Applied Spectral Imaging, Israel). Post-processing elaboration of all the pictures was made based using CorelDRAW Graphics Suite 11 (Corel Corporation, Canada). The metaphase chromosomes were karyotyped by size and position of the centromere in according to Levan et al. (1964 (link)). Metacentrics (m) and submetacentrics (sm) were classified as bi-armed, whereas subtelocentrics (st) and acrocentrics (a) as mono-armed chromosomes.