CompCytogen 10(4): 603-613 (2016) COMPARATIVE A reerrerewet open-access over

doi: 10.3897/CompCytogen.v | 04.9507 Kan Cyto genetics

http://compcytogen.pensoft.net International journal of Plant & Animal Cytogenetics,

Karyosystematics, and Molecular Systematics

An uncommon co-localization of rDNA 5S with major rDNA clusters in Callichthyidae (Siluriformes): a report case in Corydoras carlae Nijssen & Isbriicker, 1983

Rafael Henrique da Rocha', Lucas Baumgartner’, Leonardo Marcel Paiz”?,

Vladimir Pavan Margarido”?, Carlos Alexandre Fernandes’, Eder André Gubiani'

| Universidade Estadual do Oeste do Parand, Centro de Engenharias e Ciéncias Exatas, 85903-000 Toledo, Parand, Brazil2. Universidade Estadual do Oeste do Parand, Centro de Ciéncias Biolégicas e da Saude, 85819- 110 Cascavel, Paranda, Brazil 3 Universidade Estadual de Maringd, Centro de Ciéncias Biolégicas, 87020-900 Maringd, Parand, Brazil 4 Universidade Estadual de Mato Grosso do Sul, Unidade Universitaria de Mundo Novo, 79980-000 Mundo Novo, Mato Grosso do Sul, Brazil

Corresponding author: Carlos Alexandre Fernandes (fxande@gmail.com)

Academic editor: NV. Bogutskaya | Received 10 June 2016 | Accepted 12 October 2016 | Published 18 November 2016 http://zoobank.ore/ CBDEBC52-CB24-47A9-B7E3-62ED285BB9B2

Citation: Rocha RH, Baumgartner L, Paiz LM, Margarido VP, Fernandes CA, Gubiani EA (2016) An uncommon co-localization of rDNA 5S with major rDNA clusters in Callichthyidae (Siluriformes): a report case in Corydoras carlae Nijssen & Isbriicker, 1983. Comparative Cytogenetics 10(4): 603-613. doi: 10.3897/CompCytogen.v10i4.9507

Abstract

Corydoras Lacepéde, 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 karyo- typic formula were recorded for many species of this group. In current study, we provided the first cytoge- netic information of Corydoras carlae Nijssen & Isbriicker, 1983, an endemic fish species from Iguassu Riv- er basin, Parana 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 map- ping 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.

Keywords Ag-NORs, cytogenetic markers, ribosomal DNA, heterochromatin, karyotype

Copyright Rafael Henrique da Rocha et a. 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.

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Introduction

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 fluo- rescent in situ hybridization (FISH). The major rDNA sequences detected by FISH always coincided with silver-stained NORs (Ag-NORs) location, although in species with multiple Ag-NORs the number of markings was usually smaller than the regions detected by the DNA probes.

The most common condition in the karyotype of different fish groups is the po- sitioning of ribosomal genes in different chromosome pairs (Galetti Jr. and Martins 2004). However, syntenic localization of the major rDNA clusters and the 5S sites were observed for the first time in the genus Corydoras Lacepéde, 1803 (present study) and Callichthys callichthys (Linnaeus, 1758) (Konerat et al. 2014), the other integrant of the family Callichthyidae. In Loricariidae, Kronichthys lacerta (Nichols, 1919), Js- brueckerichthys duseni (Miranda Ribeiro, 1907), Parotocinclus maculicauda (Steindach- ner, 1877) and Trichomycterus sp. (Ziemniczak et al. 2012) also presented syntenic localization of ribosomal genes. Thus, the mapping of ribosomal genes has added im- portant information about the chromosomal diversification in Corydoras, as in other groups of Siluriformes.

Callichthyidae is a family of the order Siluriformes widely distributed in Neo- tropical region, which has 215 valid species, divided in two subfamilies, Callichthyinae with 17 valid species and Corydoradinae with 198 valid species (Eschmeyer and Fong 2016). Corydoras is the most specious and cytogenetically studied genus of Corydora- dinae, demonstrating different diploid numbers, which may vary from 2n = 40 chro- mosomes in C. nattereri Steindachner, 1876 (Oliveira et al. 1990, 1993) to 2n = 134 chromosomes in C. aeneus (Gill, 1858) (Turner et al. 1992).

Considering aspects related to number and morphology of chromosomes, as well as analysis of DNA content, Oliveira et al. (1992) and Shimabukuro-Dias et al. (2004) proposed the existence of five groups of species in Corydoras. However, the vast ma- jority of studies in Corydoras is restricted to conventional analysis and little is known about location of the different types of rDNA, only in C. paleatus (Jenyns, 1842) and C. ehrhardti Steindachner, 1910 for 18S rDNA (Artoni et al. 2006) and C. britskii (Nijssen & Isbriicker, 1983) for 18S and 5S rDNA (Takagui et al. 2014), making essential the development of studies with this approach to better understand the rela- tionships between species of Corydoras.

Thus, the current paper presents the first cytogenetic description of Corydoras car- lae, 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.

An uncommon co-localization of rDNA 5S with major rDNA clusters in Callichthyidae... 605

Materials and methods

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), Parana 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. 1). Voucher specimens were deposited in the fish collection of the Nucleo de Pesquisas em Limnologia Ictiologia e Aquicultura (NUPELIA), Universidade Estadual de Maringa, Parana, Brazil, as Corydoras carlae (NUP 17885).

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 Parana (License Number: Protocol 13/09 - CEEAAP/Unioeste). Before the evisceration pro- cess, the individuals were anesthetized by an overdose of clove oil (Griffiths 2000). Metaphase chromosomes were obtained from anterior kidney cells using the air-drying technique (Bertollo et al. 1978). Analysis of the C-positive heterochromatin (C-bands) followed the basic procedure of Sumner (1972), with some minor adaptations (Lui et al. 2012). The NORs were detected by means of silver nitrate staining (Ag- NORs), ac- cording to Howell and Black (1980). The chromosomes were classified as metacentric (m), submetacentric (sm), and subtelocentric (st) according to their arm ratio (Levan et al. 1964). For the determination of the fundamental number (FN), or number of chromosome arms, the m, sm and st chromosomes were considered as bearing two arms and the acrocentric chromosomes only one arm.

Figure |. Localization of Florido River from the Iguassu River basin, where Corydoras carlae individuals

were captured. Red triangle indicates the sampled point.

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The localization of the 5S and 18S rDNA sites in the chromosomes was performed using the fluorescence in situ hybridization (FISH) method (Pinkel et al. 1986) with modifications (Margarido and Moreira-Filho 2008), with probes obtained from the fish species Leporinus elongatus Valenciennes, 1850 (Martins and Galetti Jr 1999) and Prochilodus argenteus Spix & Agassiz, 1829 (Hatanaka and Galetti Jr 2004), respective- ly. The probes were labelled through nick translation, with digoxigenin-11-dUTP (5S rDNA) and biotin-16-dUTP (18S rDNA) (Roche). Detection and amplification of the hybridization signal were made using avidin-FITC and anti-avidin biotin (Sigma) for probes labelled with biotin, and anti-digoxigenin rhodamine (Roche) for probes labelled with digoxigenin. Slides were counterstained with DAPI (50 pg/mL) and ana- lysed in epifluorescence microscope (Olympus BX61). The images were captured using the software DP controller (Media Cybernetics).

Results

Corydoras carlae presented a modal diploid number of 46 chromosomes in males and females, and the karyotype contained 22 metacentric, 22 submetacentric and 2 subte- locentric chromosomes (22m+22sm+2st), yielding a FN of 92 in both sexes (Fig. 2a). The Ag-NORs was detected in the terminal position on the long arm of metacentric pair 6 (Box Fig. 2a). Positive C-band heterochromatins were detected in the centro- meric and pericentromeric regions of nine and eight pairs, respectively, and coinci- dent with the ribosomal sites (Fig. 2b). The double-FISH technique showed 5S rDNA cluster appears interspersed with 18S cistrons in the terminal portion of the long arm of pair 6 (Fig. 2c). Thus, featuring synteny and co-location of ribosomal genes in C. carlae. The ideogram summarizes all markers on chromosomes of C. carlae (Fig 3).

Discussion

Cytogenetic studies have classified the species of the genus Corydoras into five groups according to their karyotype composition (Oliveira et al. 1992, Shimabukuro-Dias et al. 2004). Corydoras carlae has been included in group 4 (2n = 40-52 chromosomes, with many metacentric and submetacentric chromosomes). Considering our results, three species of this group occurring in the Iguassu River basin were cytogenetically analyzed: C. carlae (2n=46, 22m+22sm+2st), collected in the Lower Iguassu River; C. paleatus (2n=44, 20m+24sm) collected in the Upper Iguassu River (Oliveira et al. 1993), and C. paleatus and C. ehrhardti (2n=44, 18m+26sm), collected in the Upper Tibagi River (Artoni et al. 2006).

Individuals of C. carlae analyzed here probably do not co-occur with C. palea- tus from Upper Iguassu River, since the lower portion is characterized by numerous waterfalls which gave rise to several reservoirs (Baumgartner et al. 2012). Therefore, the geographic isolation of C. carlae may have facilitated the establishment of this nu-

An uncommon co-localization of rDNA 5S with major rDNA clusters in Callichthyidae... 607

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Figure 2. Karyotypes of Corydoras carlae stained with a Giemsa b C-banded and € after double FISH with 5S rDNA probes (red) and 18S rDNA (green). The NORs bearing chromosomes (pair 6) are boxed. Bar = 10 pm.

608 Rafael Henrique da Rocha et al. / Comparative Cytogenetics 10(4): 603-613 (2016)

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Figure 3. Ideogram of Corydoras carlae, showing the heterochromatin, Ag-NORs, 18S and 5S rDNA

distribution pattern.

merical and structural karyotypic variation, as also observed in different populations of Glanidium ribeiroi Haseman, 1911 along the Iguassu River basin (Lui et al. 2015). Thus, the lack of gene flow among Corydoras species in the Iguassu River basin could favor different changes in each sample, supposedly resulting in speciation.

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 1). These data show that most species have simple NORs located in the terminal position, as in the case of C. carlae. However, not all species have this pattern, as in the case of C. simulatus Weitzman & Nijssen, 1970 with interstitial NORs (Oliveira et al. 1992), as well as C. britskii (Takagui et al. 2014), C. simulatus, Corydoras sp. Galheiro river, C. flaveolus Thering, 1911 and C. metae Eigenmann, 1914 (Oliveira et al. 1992), which exhibits a systems of mul- tiple NORs. According to Oliveira and Gosztonyi (2000), the condition of simple Ag-NORs in terminal location is the possible basal condition for Siluriformes. Thus, C. carlae and other species presenting simple Ag-NORs in terminal location seem to maintain this basal condition.

Despite exhibiting wide variation on the diploid number, chromosome morphol- ogy 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 het- erochromatic blocks (Takagui et al. 2014), which were not observed in this study.

The mapping of 18S rDNA and 5S rDNA are scarce in Callichthyidae, being known only for some species (Table 1). Concerning the genus Corydoras, C. carlae exhibited only one chromosome pair bearing 18S rDNA sites, as well as C. ehrhardti (Artoni et al. 2006), confirming the system of simple NORs evidenced by silver im- pregnation for both species. FISH with rDNA probes has helped detect the presence of inactive NORs, as in the case of C. paleatus (Artoni et al. 2006), which presented multiple NORs sites after 18S-FISH, while the silver impregnation had detected only simple NORs. Thus, studies with 18S-FISH can be useful for better evaluating the pattern distribution of the NORs in Corydoras.

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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 subtelo- centric chromosomes (Takagui et al. 2014). In C. carlae, the 5S rDNA was displayed at terminal position on the long arm of the metacentric pair 6. The presence of one chromosome pair bearing 5S rDNA is a common feature in different families of Silu- riformes (Swarca et al. 2009), although multiple loci of 5S rDNA have been observed in Callichthyinae (Table 1). Inter— and intra—individual numerical and position varia- tions of 5S rDNA cistrons have been observed in Callichthyidae and seem to represent a species-specific marker.

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 main- tained linked in the same chromosome area (Martins and Galetti Jr 1999). This may explain why most vertebrates have these sequences on different chromosomes. Inter- estingly, 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 (Vicente et al. 2001) and Astyanax paranae Eigenmann, 1914 cited as Astyanax scabripinnis (Mantovani et al. 2005), or in adjacent regions, as in Triportheus nematurus (Kner, 1858) (Diniz et al. 2009), Mugil incilis Hancock, 1830 (Hett et al. 2011), Kronichthys lacerta, Isbrueckerichthys duseni, Parotocinclus maculicauda, Trichomycterus sp. (Ziemniczak et al. 2012) and Callichthys callichthys (Konerat et al. 2014), or the 5S rDNA interspersed along the clusters of 45S rDNA (co-localization), as in Astyanax altiparanae Garutti & Britski, 2000, Astyanax lacustris (Liitken, 1875), Astyanax fasciatus (Cuvier, 1819), Astyanax schubarti Britski, 1964 and Astyanax paranae cited as Astyanax scabripinnis (Almeida-Toledo et al. 2002), Solea senegalensis Kaup, 1858 (Cross et al. 2006), Bryconamericus cf. iheringii (Piscor et al. 2013) and Corydoras carlae (present study).

Despite little studies about mapping of rDNA genes in Callichthyidae, the major- ity 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.

Acknowledgments

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 Junio da Graga and MSc. Luiz Tencatt for taxonomic identification of the specimens. We would also like to express our gratitude to Brazilian agency Funda¢ao Araucaria for scholar- ship to first author.

An uncommon co-localization of rDNA 5S with major rDNA clusters in Callichthyidae... 611

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