Heterochromatins and band karyotypes in three species of salmonids

Summary The heterochromatins of rainbow trout (Salmo gairdneri R.), brown trout (Salmo trutta fario L.) and brook trout (Salvelinus fontinalis M.) were characterized by sequential chromomycin A₃/distamycin A/DAPI (CDD) and DAPI/actinomycin D (DAPI/AmD) fluorescence. On most biarmed chromosomes, an equilocal localization of prominent DAPI/AmD positive, chromomycin A₃ negative, AT-rich blocks at the centromeres were observed in all three species. Band karyotypes of the three species were established. In rainbow trout, several DAPI/AmD positive heterochromatin blocks behaved positive in a silver-staining method. Mitotic and interphase studies proved the presence of inter-individual NOR variation in brown trout. The NORs of brook trout were localized on chromosomes 5, 10, 14, 15 and 29.     

Localisation of NORs and counterstain-enhanced fluorescence studies in Salmo gairdneri and Salmo trutta (Pisces,Salmonidae)

Summary The karyotypes of the rainbow trout (Salmo gairdneri R.) and the brown trout (Salmo trutta L.) were analyzed by means of silver staining and the chromomycin A₃/distamycin A/DAPI fluorescence banding technique. The nucleolus organizer regions (NORs) were localized at the secondary constrictions of chromosome no. 14 in S. gairdneri and of chromosome no. 10 in S. trutta. Additional silver positive dots were observed at or close to several centromeres in S. gairdneri. Brilliant chromomycin A₃ (CMA₃) fluorescence heterochromatin blocks were localized on both sides of the nucleolar constrictions in S. gairdneri. A polymorphic CMA₃ positive band was detected close to the NORs of S. trutta. No distamycin A/DAPI intense heterochromatin blocks were detected in the genomes of the two Salmo species investigated.     

Chromosome CPD(PI/DAPI)- and CMA/DAPI-Banding Patterns in Allium cepa L.

Chromosome banding patterns of Allium cepa L. were obtained by using fluorescent dye combinations chromomycin A₃ (CMA) + 4",6-diamidino-2-phenylindole (DAPI), DAPI + actinomycin D (AMD) and propidium iodide (PI) + DAPI. In A. cepa,telomeric heterochromatin displayed dull fluorescence after staining with DAPI and DAPI/AMD. After joint staining with the GC-specific CMA and AT-specific DAPI, the CMA-positive fluorescence of the NOR region and the telomeric bands of C-heterochromatin was observed. In combination with DAPI, PI, a dye with low AT/GC specificity, produced almost uniform fluorescence of chromosomal arms and heterochromatin, whereas the NOR-adjoining regions displayed bright fluorescence. Denaturation of chromosomal DNA (2 × SSC, 95°C for 1–3 min) followed by renaturation (2 × SSC, 37°C, 12 h) altered the chromosome fluorescence patterns: specific PI-positive bands appeared and the contrast of CMA-banding increased. Bright fluorescence of NOR and adjoining regions was also observed in the case. Three-minute denaturation led also to a bright PI-positive fluorescence of telomeric heterochromatin. The denaturation of chromosomal DNA before staining results in changes of the DAPI fluorescence pattern and in the appearance of bright DAPI fluorescence in GC-rich NOP regions. The mechanisms underlying the effects of denaturation/renaturation procedures on chromosome banding patterns obtained with different fluorochromes are discussed.     

Nucleolus-organizer regions and heterochromatin in three species of Bovidae

Ag-NOR staining and a counterstain-enhanced fluorescence technique (chromomycin A₃/distamycin A/DAPI staining = CDD-method) have been applied to ibex (Capra ibex L.), chamois (Rupicapra rupicapra L.) and bison (Bison bison L.) chromosomes.Chromomycin A₃ visualization led to a well defined R-banding pattern along the chromosome arms and to a clear demonstration of centric heterochromatic bands of variable size. The nucleolus organizer regions (NORs) were found in the telomeric regions of the chromosomes 2, 3, 4, 5 and 28 of the ibex, of the chromosomes 1/3 (short arm), 2, 4, 5 and 28 of the chamois and of the chromosomes 2, 3, 4, 11 and 28 of the bison.     

Chromosome banding study of European catfish,Silurus glanis (Pisces,Siluridae)

The karyotype of European catfish (Silurus glanis L.) was analyzed sequentially by means of silver staining and the chromomycin A₃ (CMA₃)/distamycin A (DA)/DAPI fluorescence technique and by C-banding, respectively. The nucleolus organizer regions (NORs) were localized on the submetacentric pair No. 14. Brilliant CMA₃ fluorescent heterochromatin blocks corresponded to the NORs visualized by silver staining. No DA/DAPI-bright positive fluorescent patterns were detected while C-banding led to the detection of specific banding patterns on several chromosome pairs.—Using these banding data, the karyotype of S. glanis was redescribed.     

Reverse fluorescent chromosome banding with chromomycin and DAPI

Two DNA binding guanine-specific antibiotics, chromomycin A₃ (CMA) and the closely related mithramycin (MM), were used as chromosome fluorescent dyes. Root-tip metaphase chromosomes of three plant species and human metaphase chromosomes were sequentially stained with CMA or MM and the DNA binding AT-specific fluorochrome 4-6-diamidino-2-phenylindole (DAPI). In some cases a non-fluorescent counterstain was used as contrasting agent: methyl green in conjunction with CMA, and actinomycin D (AMD) in combination with DAPI. — In all three plant species, Vicia faba, Scilla siberica, and Ornithogalum caudatum, the nucleolus organiser regions and/or associated heterochromatin displayed very bright fluorescence with CMA and MM and, in general, heterochromatic segments (C-bands) which were bright with CMA and MM were pale with DAPI whereas segments which were dim with CMA and MM displayed very bright fluorescence with DAPI. — Human metaphase chromosomes showed a small longitudinal differentiation in CMA fluorescence, which was essentially the reverse of the banding pattern obtained with AMD/DAPI double-staining, but of lower contrast. The CMA-banding pattern appears to be similar to the pattern found by R-banding procedures.     

Characterization of heterochromatin by sequential counterstain-enhanced fluorescence in three domestic bird species: Meleagris gallopavo, Columba livia domestica, and Anser anser L

The karyotypes of three avian species--Meleagris gallopavo, Anser anser L., and Columba livia domestica--were investigated by means of counterstain-enhanced fluorescence techniques (chromomycin A3/distamycin A/DAPI followed by DAPI/actinomycin D staining). A heterochromatin characterization of macro- and microchromosomes was performed. CMA3-positive (GC-rich) regions in the turkey included the telomeres of chromosomes 1, 3, 4, and Z. In the goose, the chromosome 2 was also CMA3-bright at the telomeres. The W chromosome possessed large amounts of CMA3-bright material on the short arm in both the turkey and the goose. Two types of centromeric heterochromatin were distinguished on acro- to telocentric chromosomes 6 to 14 in the pigeon. The microchromosomal heterochromation of the turkey and goose was GC-rich but had a high degree of variation. In the pigeon, several microchromosomes possessed predominantly AT-rich heterochromatin.     

Nucleolus organizer regions and heterochromatin in the zebu (Bos indicus L.)

Summary Ag-NOR staining and a counterstain enhanced fluorescence technique (chromomycin A₃/distamycin A/DAPI-staining = CDD-method) and G-banding, respectively, have been applied to the zebu (Bos indicus L.) chromosomes. The nucleolus organizer regions (NORs) were found in the telomeric regions of chromosomes nos. 2, 3, 4, 11, and 28. CDD staining led to a well-defined R-banding pattern along the chromosome arms and to the visualization of centric heterochromatic bands of variable sizes.     

Chromosome banding and FISH with rDNA probe in the diploid and tetraploid loach Misgurnus anguillicaudatus

The chromosomes of the diploid and tetraploid loach Misgurnus anguillicaudatus were analyzed by staining with Ag, chromomycin A₃ (CMA₃)/distamycin A (DA), and DA/4′,6-diamidino-2-phenylindole (DAPI), and using fluorescence in situ hybridization (FISH) with 5.8S + 28S rDNA as a probe. Nucleolus organizer regions (NORs) were mapped to the telomeric region of the short arms of the largest (first) metacentric chromosome pair in the diploid loach with 2n = 50 and the homologous quartet in the tetraploid loach with 4n = 100. The NORs were positive at the same region of the first metacentric chromosome for Ag and CMA₃/DA stainings, but negative for DA/DAPI staining. Four signals at the homologs within the same quartet suggest the duplication of the entire genome from diploid to tetraploid status. However, a size difference was detected between the rDNA signals by FISH and CMA₃ banding.     

Characterization of the canine karyotype by counterstain-enhanced chromosome banding

The application of the counterstain-contrasted fluorescent banding technique to canine chromosomes provided an improved capability to highlight specific heterochromatic regions and to produce well defined banding patterns both on mitotic and meiotic chromosomes. Triple staining with chromomycin A3 - distamycin A - DAPI revealed the occurrence of DA - DAPI positive heterochromatin in chromosomes 33, 36, 37, and 38. Pachytene nuclei present more favourable material for the detection of very small amounts of DA - DAPI material than mitotic division stages. Counterstain-enhanced chromomycin R-banding greatly facilitated chromosome identification. A standard R-band karyotype of Canis familiaris is proposed and described in some detail. DAPI - actinomycin D staining produced a QFH-type banding pattern and enhanced differentiation of some polymorphic regions.     

Chromosome CPD(PI/DAPI)- and CMA/DAPI-banding patterns in Allium cepa L

Chromosome banding patterns of Allium cepa L. were obtained by using fluorochrome combinations chromomycin A3 (CMA) + 4',6-diamidino-2-phenylindole (DAPI), DAPI + actinomycin D (AMD) and propidium iodide (PI) + DAPI. In A. cepa, telomeric heterochromatin displayed dull fluorescence after staining with DAPI and DAPI/AMD. After staining with the GC-specific CMA and AT-specific DAPI, the CMA-positive fluorescence of the NOR region and the telomeric bands of C-heterochromatin was observed. In combination with DAPI, PI, a dye with low AT/GC specificity, produced almost uniform fluorescence of chromosomal arms and heterochromatin, whereas the NOR-adjoining regions displayed bright fluorescence. Denaturation of chromosomal DNA (95 degrees C for 1-3 min) followed by renaturation in the 2 x SSC buffer (37 degrees C, 12 h) altered the chromosome fluorescence patterns: specific PI-positive bands appeared and the contrast of CMA-banding increased. Bright fluorescence of the NOR and adjoining regions was also observed in the case. Three-minute denaturation led also to a bright PI-positive fluorescence of telomeric heterochromatin. The denaturation of chromosomal DNA before staining results in changes of the DAPI fluorescence pattern and in the appearance of DAPI fluorescence in GR-rich NOP regions. The mechanisms underlying the effects of denaturation/renaturation procedures on chromosome banding patterns obtained with different fluorochromes are discussed.     

Chromosome maps of the plant family Trilliaceae: nucleotide composition of heterochromatin an localization of 18S-26S rRNA-genes of four-leafed paris (Paris quadrifolia L.)

Using nucleotide-specific agents Hoechst 33258, actinomycin D, chromomycin A3, and distamycin A, the Paris quadrifolia L. karyotype, and the location and nucleotide composition of heterochromatic bands were studied. The chromosome ideogram of H33258/AMD and CMA/DA heterochromatic bands was created by an image analysis system with the Videotest-Kario software. By fluorescence in situ hybridization, the 18S and 26S rRNA genes were mapped.     

Karyotype analysis and heterochromatin differentiation with Giemsa C-banding and fluorescent counterstaining inCephalanthera (Orchidaceae)

Detailed studies of the chromosomes of the three Austrian species of the genusCephalanthera showed them all to have basically similar karyotypes. BothC. damasonium (2n = 36) andC. longifolia (2n = 32) have three large and several classes of smaller chromosome pairs. The karyotype ofC. rubra (2n = 44) is composed of four large and several groups of smaller pairs. The heterochromatin in these species amounts to about 10% of total karyotype length. All the chromosomes have Giemsa-positive centromeres, but only a few have intercalary or terminal bands. Using differential fluorescent staining with DAPI/actinomycin D, quinacrine/actinomycin D (both A-T specific), and chromomycin A₃/distamycin A (G-C specific) three different types of major heterochromatic bands can be characterized in respect of their satellite DNA composition: highly A-T rich, slightly A-T rich, and very G-C rich. The chromosomes ofC. longifolia contain more A-T rich C-bands than those ofC. damasonium, while the latter's have more G-C rich heterochromatin. In both species several C-bands appear as secondary constrictions or gaps in the Feulgen-stained chromosomes, but most likely, in each species there is only one pair of chromosomes where the secondary constrictions function as nucleolus organizing regions. No major intraspecific variation could be observed except on one small chromosome pair ofC. longifolia which had a heteromorphic C-band in most individuals. Possible pathways of karyotype evolution involving polyploidy and Robertsonian events are discussed.     

The organization of DNA in the mitotic and polytene chromosomes of Sciara coprophila

The organization of DNA in the mitotic metaphase and polytene chromosomes of the fungus gnat, Sciara coprophila, has been studied using base-specific DNA ligands, including anti-nucleoside antibodies. The DNA of metaphase and polytene chromosomes reacts with AT-specific probes (quinacrine, DAPI, Hoechst 33258 and anti-adenosine) and to a somewhat lesser extent with GC-specific probes (mithramycin, chromomycin A₃ and anticytidine). In virtually every band of the polytene chromosomes chromomycin A₃ fluorescence is almost totally quenched by counterstaining with the AT-specific ligand methyl green. This indicates that GC base pairs in most bands are closely interspersed with AT base pairs. The only exceptions are band IV-8A3 and the nucleolus organizer on the X. In contrast, quinacrine and DAPI fluorescence in every band is only slightly quenched by counterstaining with the GC-specific ligand actinomycin D. Thus, each band contains a moderate proportion of AT-rich DNA sequences with few interspersed GC base pairs. — The C-bands in mitotic and polytene chromosomes can be visualized by Giemsa staining after differential extraction of DNA and those in polytene chromosomes by the use of base-specific fluorochromes or antibodies without prior extraction of DNA. C-bands are located in the centromeric region of every chromosome, and the telomeric region of some. The C-bands in the polytene chromosomes contain AT-rich DNA sequences without closely interspered GC base pairs and lack relatively GC-rich sequences. However, one C-band in the centromeric region of chromosome IV contains relatively GC-rich sequences with closely interspersed AT base pairs. — C-bands make up less than 1% of polytene chromosomes compared to nearly 20% of mitotic metaphase chromosomes. The C-bands in polytene chromosomes are detectable with AT-specific or GC-specific probes while those in metaphase chromosomes are not. Thus, during polytenization there is selective replication of highly AT-rich and relatively GC-rich sequences and underreplication of the remainder of the DNA sequences in the constitutive heterochromatin.     

Supernumerary heterochromatic segments associated with the nucleolar chromosomes of Pyrgomorpha conica (Orthoptera) contain methylated rDNA sequences

The two nucleolus organizing chromosome pairs of the grasshopper Pyrgomorpha conica can carry a proximal supernumerary heterochromatic segment. We employed different cytological techniques to characterize and analyze the possible origin of this segment. The supernumerary segment and the nucleolus organizing regions (NORs) show similar responses after C-banding plus either Giemsa or acridine orange, and chromomycin A₃/distamycin A staining to detect GC-rich chromosome regions. Fluorescence in situ hybridization with a biotinylated rDNA probe demonstrated that the segment originated by amplification of the rDNA genes. However, as the silver staining indicates, the ribosomal genes present in the segment are not active since no nucleolus is formed. The use of in situ digestion with the isoschizomeric MspI and HpaII restriction endonucleases and subsequent Giemsa, ethidium bromide or chromomycin A₃/distamycin A staining, suggests that the segment has been inactivated by DNA methylation.     

Growth,Reproduction and Condition of Roach (Rutilus rutilus (L.)), Perch (Perca fluviatilis,L.) and Ruffe (Gymnocephalus cernuus (L.)) in Eutrophic Lake Aydat (France)

The growth, reproduction and condition of adults of the three dominant fish species (roach, Rutilus rutilus, (L.); perch, Perca fluviatilis, L. and ruffe, Gymnocephalus cernuus, (L.)) in the eutrophic Lake Aydat were studied over one year cycle. Compared to published data, the growth of R. rutilus and G. cernuus was about average, while that of P. fluviatilis was below average. Comparing to literature, the fecundity of R. rutilus and G. cernuus was low but their oocytes were large. In contrast, P. fluviatilis had a high fecundity but small oocytes. At the end of summer, an abrupt decrease in the condition was recorded only for perch, probably due to stress as a result of environmental conditions. The sex-ratio was in favour of females for the three studied species but, in contrast to G. cernuus, the sex-ratio of R. rutilus and P. fluviatilis increased significantly also with age. It is concluded that Lake Aydat is a more favourable environment for R. rutilus and G. cernuus than for P. fluviatilis.     

Adaptive major histocompatibility complex (MHC) and neutral genetic variation in two native Baltic Sea fishes (perch Perca fluviatilis and zander Sander lucioperca) with comparisons to an introduced and disease susceptible population in Australia (P. fluviatilis): assessing the risk of disease epidemics

This study assessed the major histocompatibility complex (MHC) and neutral genetic variation and structure in two percid species, perch Perca fluviatilis and zander Sander lucioperca, in a unique brackish ecosystem, the Baltic Sea. In addition, to assess the importance of MHC diversity to disease susceptibility in these populations, comparisons were made to an introduced, disease susceptible, P. fluviatilis population in Australia. Eighty‐three MHC class II B exon 2 variants were amplified: 71 variants from 92 P. fluviatilis samples, and 12 variants from 82 S. lucioperca samples. Microsatellite and MHC data revealed strong spatial genetic structure in S. lucioperca, but not P. fluviatilis, across the Baltic Sea. Both microsatellite and MHC data showed higher levels of genetic diversity in P. fluviatilis from the Baltic Sea compared to Australia, which may have facilitated the spread of an endemic virus, EHNV in the Australian population. The relatively high levels of genetic variation in the Baltic Sea populations, together with spatial genetic structure, however, suggest that there currently seems to be little risk of disease epidemics in this system. To ensure this remains the case in the face of ongoing environmental changes, fisheries and habitat disturbance, the conservation of local‐scale genetic variation is recommended.     

Development of 55 novel polymorphic microsatellite loci for the critically endangered Zingel asper L. (Actinopterygii: Perciformes: Percidae) and cross-species amplification in five other percids

By combining biotin-enrichment protocol and next generation pyrosequencing, through 454 GS-FLX Titanium technology, 55 polymorphic microsatellites loci with perfect motif were isolated from the Rhone streber (Zingel asper), a critically endangered European fish species. Eight multiplex PCR kits were optimised in order to genotype a total of 58 polymorphic loci, including three previously published loci. The level of genetic diversity was assessed for 68 Z. asper, 30 Sander lucioperca, 33 Perca fluviatilis and four Gymnocephalus schraetzer individuals. Amplification success was also assessed on Romanichthys valsanicola and Zingel streber using single individuals. These markers will be useful to investigate the population structure of the highly fragmented Rhone streber. They represent a powerful tool for conservation issues and evolutionary approaches of this endemic species. Moreover, part of our markers demonstrated applicability to other percid species, allowing for potential applications to fisheries and aquaculture management.     

Feeding habits and ontogenic changes in digestive enzyme patterns in five freshwater teleosts

Feeding habits and the activity of digestive enzymes (total alkaline proteases, α‐amylase and lipase) from dace Leuciscus leuciscus, roach Rutilus rutilus, Prussian carp Carassius auratus gibelio, perch Perca fluviatilis and pikeperch Sander lucioperca fry were studied in the Malye Chany Lake–Kargat Estuary (western Siberia, Russia). The diet of fry from all studied species was mainly composed of chironomid larvae and zooplanktonic organisms (i.e. cladocera and copepoda), whereas carnivorous species such as P. fluviatilis and S. lucioperca also preyed on fry from other fishes while detritus and microalgae were also important in the diet of ommivorous species. When comparing diet similarity (Sørensen–Dice index, Q_S) among fry at different stages of development, both omnivorous and carnivorous species showed a high level of similarity (0·67 < Q_S < 0·89 and 0·73 < Q_S < 0·89, respectively). Diet similarity values were in agreement with the overall digestive activity profile analysed by cluster analysis. Diet similarity suggested potential trophic competition when zooplanktonic and benthic prey began to decline towards autumn. The analysis of pancreatic digestive enzymes revealed a correlation among their activities and fry feeding habits with α‐amylase:total proteases (A:P) values higher than 1 in omnivorous species and lower (A:P ≤ 1) in carnivorous species.     

Cold-sensitive chromosome regions and heterochromatin inCestrum aurantiacum (Solanaceae)

The karyotype ofCestrum aurantiacum was analyzed for the presence of coldsensitive regions (CSRs) and other types of constitutive heterochromatin. A range of techniques was employed including the fluorescent DAPI, chromomycin/DAPI double staining and actinomycin D/DAPI counter-staining, and the non-fluorescent C-banding applied as single or sequential staining, sequential N-banding and silver impregnation. Four classes of constitutive heterochromatin were recognized: CSRs, nucleolar organizers, non-nucleolar chromomycin-positive bands, and indifferently fluorescent bands. The banded karyotype ofC. aurantiacum is compared with those of otherCestrum species. The sectionsHabrothamnus andCestrum are not karyologically distinct.