Research Article |
Corresponding author: Carlos Alexandre Fernandes ( fxande@gmail.com ) Academic editor: Nina Bogutskaya
© 2016 Rafael Henrique da Rocha, Lucas Baumgätner, Leonardo Marcel Paiz, Vladimir Pavan Margarido, Carlos Alexandre Fernandes, Eder Andre Gubiani.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Rocha RH, Baumgärtner L, Paiz LM, Margarido VP, Fernandes CA, Gubiani ÉA (2016) An uncommon co-localization of rDNA 5S with major rDNA clusters in Callichthyidae (Siluriformes): a report case in Corydoras carlae Nijssen & Isbrücker, 1983. Comparative Cytogenetics 10(4): 603-613. https://doi.org/10.3897/CompCytogen.v10i4.9507
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Corydoras Lacepède, 1803 is the most specious genus of Corydoradinae subfamily and many of its species are still unknown in relation to molecular cytogenetic markers. However, the diploid number and karyotypic formula were recorded for many species of this group. In current study, we provided the first cytogenetic information of Corydoras carlae Nijssen & Isbrücker, 1983, an endemic fish species from Iguassu River basin, Paraná State, Brazil. The individuals were collected in Florido River, a tributary of Iguassu River and analysed with respect to diploid number, heterochromatin distribution pattern, Ag-NORs and mapping of 5S and 18S ribosomal genes. The karyotype of this species comprises 46 chromosomes arranged in 22m+22sm+2st. The heterochromatin is distributed in centromeric and pericentromeric positions in most of the chromosomes, and also associated with NORs. The Ag-NORs were detected in the terminal position on the long arm of the metacentric pair 6. The double-FISH technique showed that 5S rDNA and 18S rDNA were co-localized in the terminal portion on the long arm of the metacentric pair 6. This condition of co-localization of ribosomal genes in C. carlae seems to represent a marker for this species.
Ag-NORs, cytogenetic markers, ribosomal DNA, heterochromatin, karyotype
In higher eukaryotes, rDNA is organized into two distinct gene classes: major class (45S rDNA), which contains the genes that code for the 18S, 5.8S and 28S rRNAs, and the minor class (5S rDNA), which contains the genes that code for 5S rRNA. Fish species have been analyzed for 5S and 18S rDNA location in chromosomes using fluorescent
The most common condition in the karyotype of different fish groups is the positioning of ribosomal genes in different chromosome pairs (
Callichthyidae is a family of the order Siluriformes widely distributed in Neotropical region, which has 215 valid species, divided in two subfamilies, Callichthyinae with 17 valid species and Corydoradinae with 198 valid species (
Considering aspects related to number and morphology of chromosomes, as well as analysis of DNA content,
Thus, the current paper presents the first cytogenetic description of Corydoras carlae, focusing on karyotype characterization, heterochromatin distribution pattern and location of 5S and 18S rDNA sites. Besides the new data for the species, this study also reveal for the first time the co-localization of 5S and major rDNA in Callichthyidae.
Ten individuals (four females and six males) of Corydoras carlae were sampled in the Florido River (26°00'32.60"S; 53°25'50.70"W), Paraná State, Brazil. A sub-tributary of left margin of Lower Iguassu River that flows into Capanema River, which flows immediately above of the Iguassu falls (Fig.
Localization of Florido River from the Iguassu River basin, where Corydoras carlae individuals were captured. Red triangle indicates the sampled point.
This study was carried out in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals, approved by the Committee on the Ethics of Animal Experiments of the Universidade Estadual do Oeste do Paraná (License Number: Protocol 13/09 – CEEAAP/Unioeste). Before the evisceration process, the individuals were anesthetized by an overdose of clove oil (
The localization of the 5S and 18S rDNA sites in the chromosomes was performed using the fluorescence in situ hybridization (FISH) method (
Corydoras carlae presented a modal diploid number of 46 chromosomes in males and females, and the karyotype contained 22 metacentric, 22 submetacentric and 2 subtelocentric chromosomes (22m+22sm+2st), yielding a FN of 92 in both sexes (Fig.
Karyotypes of Corydoras carlae stained with a Giemsa b C-banded and c after double FISH with 5S rDNA probes (red) and 18S rDNA (green). The NORs bearing chromosomes (pair 6) are boxed. Bar = 10 µm.
Cytogenetic studies have classified the species of the genus Corydoras into five groups according to their karyotype composition (
Individuals of C. carlae analyzed here probably do not co-occur with C. paleatus from Upper Iguassu River, since the lower portion is characterized by numerous waterfalls which gave rise to several reservoirs (
The number and position of NORs in Corydoras species are quite variable and almost all information pertaining to the characterization of NORs in this species is based on silver nitrate impregnation (Table
Ag-NORs, major and minor ribosomal genes sites distribution in Callichthyidae. The 45S and 5S columns report the number of chromosomes bearing the cistrons and its location.
Family Callichthyidae | Locality | 2n | Ag-NOR | 45S | 5S | Note | References |
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Subfamily Corydoradinae | |||||||
Corydoras carlae | Florido River/Paraná State, Brazil | 46 | simple | 2, q terminal | 2, q terminal | Synteny, Co-localization | Present study |
Corydoras britskii | Miranda River/ Mato Grosso do Sul State, Brazil | 90 | multiple | 3, p terminal | 2 p, interstitial | Non-Synteny |
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Corydoras paleatus | Tibagi River/Paraná State, Brazil | 44 | simple | 3, q terminal | ------- | --------- |
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Corydoras ehrhardti | Tibagi River/Paraná State, Brazil | 44 | simple | 2, q terminal | ------- | --------- |
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Corydoras sodalis | from aquarium | 74 | simple | --------- | --------- | --------- |
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Corydoras arcuatus | Tabatinga River/frontier Brazil and Peru | 46 | simple | --------- | --------- | --------- |
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Corydoras trilineatus | Caripi River/Pará State, Brazil | 46 | simple | --------- | --------- | --------- |
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Corydoras schwartzi | Negro River/Amazonas State, Brazil | 46 | simple | --------- | --------- | --------- |
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Corydoras cf. simulatus | Colombia | 62 | simple | --------- | --------- | --------- |
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Corydoras sp. Caripi River | Caripi River/Pará State, Brazil | 60 | simple | --------- | --------- | --------- |
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Corydoras reticulatus | Negro River/Amazonas State, Brazil | 74 | simple | --------- | --------- | --------- |
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Corydoras aff. punctatus Negro River | Negro River/Amazonas State, Brazil | 102 | simple | --------- | --------- | --------- |
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Corydoras simulatus | Colombia | 62 | multiple | --------- | --------- | --------- |
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Corydoras sp. Galheiro River | Galheiro River/Minas Gerais State, Brazil | 84 | multiple | --------- | --------- | --------- |
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Corydoras flaveolus | Alambari River/São Paulo State, Brazil | 58 | multiple | --------- | --------- | --------- |
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Corydoras metae | Colombia | 92 | multiple | --------- | --------- | --------- |
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Subfamily Callichthyinae | |||||||
Hoplosternum littorale | Contas River/Bahia State, Brazil | 60 | simple | 2, p terminal | 4, p terminal | Non-Synteny |
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Callichthys callichthys | Paraná River/Paraná State, Brazil | 56 | simple | 2-3, p terminal e interstitial | 7-9, p interstitial and terminal | Synteny, Adjacent regions |
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Hoplosternum littorale | Coastal River/São Paulo State Brazil | 60 | simple | 2, p terminal | 4 p terminal | Non-Synteny |
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Callichthys callichthys | Contas River/Bahia State, Brazil | 54 | multiple | 7, p terminal, 5, q terminal, 1 p interstitial | 8-12, p interstitial and terminal | Non-Synteny |
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Lepthoplosternum pectorale | Paraná River/Paraná State, Brazil | 64 | multiple | 10, p terminal; 2, q terminal | 6, p terminal | Non-Synteny |
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Despite exhibiting wide variation on the diploid number, chromosome morphology and location of NORs, Corydoras species share a heterochromatin distribution pattern very similar, preferably centromeric and pericentomeric, and in most cases, associated to NORs. In C. carlae, this pattern was also observed, with heterochromatic blocks also displayed in many chromosomes. Corydoras britskii from Miranda River also showed large amount of pericentromeric heterochromatin, but with terminal heterochromatic blocks (
The mapping of 18S rDNA and 5S rDNA are scarce in Callichthyidae, being known only for some species (Table
In Corydoras, data on the location and number of 5S rDNA cistrons had only been described for C. britskii, for which was detected interstitially in a pair of subtelocentric chromosomes (
Furthermore, 5S rDNA cluster appears interspersed with 18S cistrons, featuring synteny and co-location of ribosomal genes in C. carlae. The synteny is an unusual feature in fish, and such condition could influence an unwanted translocation of 5S sequences within 45S clusters, which could probably occur if these clusters were maintained linked in the same chromosome area (Martins and Galetti Jr 1999). This may explain why most vertebrates have these sequences on different chromosomes. Interestingly, all the possible syntenic conditions have been found in fishes, both sets of genes in distinct and disjoint chromosomal regions, as observed in Parodon nasus Kner, 1859 cited as Parodon tortuosus (
Despite little studies about mapping of rDNA genes in Callichthyidae, the majority of the species share the condition of non-synteny between the 5S rDNA and 45S rDNA. Therefore, this condition of co-localization of ribosomal genes in C. carlae seems to represent a marker for this species.
The authors thank the Ineo/Gerpel, specially Poliana, Guido, Angélica and Werike by assistance in sample collection. The authors are also grateful to Dr. Weferson Júnio da Graça and MSc. Luiz Tencatt for taxonomic identification of the specimens. We would also like to express our gratitude to Brazilian agency Fundação Araucária for scholarship to first author.