Chromosome spreading of the 5S rDNA clusters in the karyotype of Gymnotus inaequilabiatus (Gymnotiformes: Gymnotidae): insights into the role of the Rex-1 and Rex-3 retrotransposable elements of this process

Lucas Pietro Ferrari Gianini¹, Ligia Carla Balini², Fernanda Errero Porto³, Luciana Andreia Borin-Carvalho² and Carlos Alexandre Fernandes^1,4

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Abstract​

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Introduction​

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The Gymnotidae (Gymnotiformes) is a monophyletic group of Neotropical freshwater fish that includes the genera Electrophorus Gill, 1864 and Gymnotus Linnaeus, 1758. These genera are distinguished by their capability to generate single-phase electric fields with variable discharges ranging from 100 to 780 Hz, primarily for purposes of localization and communication (Nelson et al., 2016). Gymnotus is particularly diverse, consisting of at least 49 species distributed across various Central and South American basins (Fricke et al., 2024). From a cytogenetic perspective, several Gymnotus species remain uncharacterized in terms of karyotyping, while some, such as G. inaequilabiatus (Valenciennes, 1839), have only received limited attention regarding their macro- and micro-karyotypic structures (Fernandes-Matioli et al., 1998; Scacchetti et al., 2011).

The Gymnotus exhibits considerable chromosomal variation among species and populations, likely due to their low vagility, which limits migration and leads to the formation of isolated populations. This variation includes differences in diploid numbers (2n), ranging from 2n = 34 in G. capanema Milhomem, Crampton, Pierczeka, Shetka, Silva & Nagamachi, 2012 (Milhomem et al., 2012) to 2n = 54 in G. carapo Linnaeus, 1758 (Foresti et al., 1984), G. mamiraua Albert & Crampton, 2001 (Milhomem et al., 2007), G. inaequilabiatus (Scacchetti et al., 2011), and G. paraguensis Albert & Crampton, 2003 (Margarido et al., 2007; Braga et al., 2021). Additionally, there are variations in the karyotype structure and the location of repetitive sequences (Fernandes-Matioli et al., 1998; Scacchetti et al., 2011; Milhomem et al., 2011, 2013; Sousa et al., 2017). Notably, some species within this genus possess multiple sex chromosome systems, exemplified by G. pantanal Fernandes, Albert, Daniel-Silva, Lopes, Crampton & Almeida-Toledo, 2005 with X₁X₁X₂X₂/X₁X₂Y (Silva, Margarido, 2005) and G. bahianus Campos-da-Paz & Costa, 1996, which features an XX/XY₁Y₂ system (Almeida et al., 2015). The pattern of constitutive heterochromatin displays a high level of intraspecific conservation among populations from the same location, as do the nucleolar organizing regions. Consequently, employing molecular markers to differentiate individuals may offer further insights into the phylogeny, which has remained ambiguous thus far (Foresti et al., 1984; Fernandes-Matioli et al., 1998; Milhomem et al., 2007).

In the Gymnotus carapo species complex, which includes G. carapo, G. sylvius Albert & Fernandes-Matioli, 1999, G. mamiraua, G. inaequilabiatus, G. paraguensis, G. capanema, and G. arapaima Albert & Crampton, 2001, the nucleolar organizer regions (NORs) are identified on only one chromosome pair, indicating a simple NOR system. Fluorescence in situ hybridization studies mostly align with 18S rDNA labeling for these species (Scacchetti et al., 2011; Milhomem et al., 2011, 2012). However, there are exceptions within other Gymnotus species that exhibit multiple NOR systems, such as G. pantanal (Fernandes, Matioli, 2005), G. coatesi LaMonte, 1935 (Machado et al., 2017), and G. jonasi Albert & Crampton, 2001 (Milhomem et al., 2011). Moreover, a greater number of chromosomes displaying 18S rDNA markings have been observed in G. coatesi and G. jonasi (Milhomem et al., 2011; Milhomem et al., 2013; Machado et al., 2017).

The 5S rDNA regions can be found in various chromosome pairs, depending on the species examined and the different collection sites within the Brazilian basins. Multiple 5S rDNA sites have been identified in species such as G. inaequilabiatus, G. cf. carapo (Scacchetti et al., 2011), and G. paraguensis (Silva et al., 2011; Sousa et al., 2017). Conversely, in certain Gymnotus species, these sites appear on fewer chromosomes, with evidence of being located on a single pair in G. sylvius (Scacchetti et al., 2011) and G. bahianus (Almeida et al., 2015) or on two pairs in G. pantanal (Silva et al., 2011) and G. pantherinus (Steindachner, 1908) (Scacchetti et al., 2011). Transposable elements (TEs) may contribute to the numerical variation observed in 5S rDNA sites; however, only some studies have used TEs in Gymnotus to test this hypothesis (Silva et al., 2016).

The TEs have been utilized as chromosomal markers in fish cytogenetics, offering valuable insights into the mechanisms of chromosome dispersion. TEs are classified into retrotransposons (Class I) and DNA transposons (Class II) based on their transposition mechanisms, which involve either an RNA intermediate or DNA, respectively (Biscotti et al., 2015; Carducci et al., 2018). According to studies by Volff et al. (1999, 2000, 2001), teleost genomes contain a wealth of retroelements, including Rex-1, Rex-3, and Rex-6, as well as other transposable elements (Ferreira et al., 2011; Silva et al., 2016; Glugoski et al., 2018; Piscor et al., 2020, among others). However, there is a notable lack of research on the Rex-1 and Rex-3 retroelements in Gymnotus.

Our research expands the existing cytogenetic data for Gymnotus by contributing insights into the Rex-1 and Rex-3 retroelements, as well as other chromosomal characteristics of G. inaequilabiatus from the upper Paraná River basin. Our findings uncover a novel instance of chromosome spreading of the 5S rDNA clusters in Gymnotus, which may be linked to the synteny of Rex-1 and Rex-3 elements with chromosomes containing 5S rDNA.

Material and methods

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Study and sampling area. A total of thirty-two (2 males and 30 females) individuals of G. inaequilabiatus (Gymnotiformes, Gymnotidae) were obtained from Riacho Água do Ó, located in the municipality of Santa Fé, Paraná, Brazil (23°01’08”S 51°51’37.8”W). Voucher specimens were deposited in the fish collection of the Universidade Estadual de Maringá, Maringá, Paraná, Brazil, as G. inaequilabiatus (NUP 6458).

Cytogenetic analysis. Mitotic chromosomes were obtained from cells extracted from the kidney following the methodology described by Bertollo et al. (2015), which consists of inhibiting the mitotic spindle fibers by applying Colchicine (0.05%) to the animal, followed by hypotonization of the cells with potassium chloride (0.075M), fixing the cell suspension with methanol and acetic acid (3:1) and finally dripping this suspension onto slides and staining with Giemsa (5%). The chromosomes were classified as metacentric (m), submetacentric (sm), subtelocentric (st), and acrocentric (a) according to (Levan et al., 1964). The fundamental number (FN) was calculated according to the chromosomal arm numbers (the chromosomes m, sm, and st were considered to contain two arms – p and q arms – and the a with one arm – only q arm). The C-banding technique described by Sumner (1972) was used to analyze constitutive heterochromatin and the slides were stained with propidium iodide (Lui et al., 2012). Ag-NORs were detected by silver nitrate (AgNO3) staining following the method described by Howell, Black (1980).

The location of the 5S and 18S rDNA sites in the chromosomes was performed by fluorescence in situ hybridization (FISH) with modifications, about high stringency condition of 77% (200 ng of each probe, 50% formamide, 10% dextran sulfate, 2xSSC, pH 7.0–7.2, at 37^oC overnight) (Pinkel et al., 1986; Margarido, Moreira-filho, 2008) using probes from the genome of Megaleporinus elongatus (Valenciennes, 1850) (Martins, Galetti Jr., 1999) and Prochilodus argenteus Spix & Agassiz, 1829 (Hatanaka, Galetti Jr., 2004), respectively. The probes were labeled through nick translation with digoxigenin-11-dUTP (5S rDNA) and biotin-16-dUTP (18S rDNA) (Roche). Detection and amplification of the hybridization signal were carried out using avidin-FITC and anti-avidin biotin (Sigma) for probes labeled with biotin and anti-digoxigenin rhodamine (Roche) for probes labeled with digoxigenin. Chromosomes were counterstained with DAPI (50 μg ml⁻¹).

Transposable element probes were produced using the primers Rex-3 [Foward (5’- CGGTGAYAAAGGGCAGCCCTG-3’) and Reverse (5’-TGGCAGACNGGGGTGGTGGT-3’) (Volff, 2006). REX-1 [Foward (5’- TTCTCCAGTGCCTTCAACACC-3’) and Reverse (5’ – TCCCTCAGCAGAAAGAGTCTGCTC-3’) (Volff et al., 1999). Amplification was performed using PCR, and the probes were labeled according to the nick translation method using the Anti-digoxigenin-Rhodamine Kit (Roche). Chromosomes were counterstained with DAPI (50 μg ml−1).

Conventional and fluorescence chromosome preparations were analyzed under an epifluorescence microscope (Olympus BX51). The images were captured using the DP controller (Media Cybernetics) software and the image composition with Adobe Photoshop CS6.

Results​

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All 32 individuals of G. inaequilabiatus exhibited a diploid chromosome number of 54, with a karyotype consisting of 34m + 18sm + 2a, with NF = 106 for both sexes (Fig. 1A). Notably, no heteromorphic sex chromosomes were detected. Silver nitrate impregnation analysis revealed that Ag-NORs were situated in the first pair of metacentric chromosomes, located in the interstitial region of the short arms (Box in Fig. 1A), coinciding with the secondary constriction.

Utilizing the C-banding technique, it was found that blocks of constitutive heterochromatin were present in most chromosomes, with their distribution primarily concentrated in the centromeric and pericentromeric regions. An exception was noted in chromosome pair 1, which displayed a continuous heterochromatic block on the short arm of both homologs (Fig. 1B).

FIGURE 1| Karyotypes of Gymnotus inaequilabiatus stained with Giemsa (A), C-banding (B), karyotypes after double FISH with 18S rDNA (in green) and 5S rDNA (in red) probes (C), FISH with Rex-1 (D) and Rex-3 (E) retroelements. The highlighted box contains the pair carrying the nucleolus organizing region after impregnation with silver nitrate. Scale bar = 10 µm.

The FISH technique, utilizing the 18S rDNA probe, affirmed the labeling results obtained through silver nitrate and did not reveal any additional inactive major ribosomal clusters (Fig. 1C). A total of 5S rDNA sites were identified on 31 chromosomes, predominantly situated in the pericentromeric regions of pairs 2, 4, 6, 11, 12, 13, 15, 16, 17, 18, 22, 24, and 27, with pair 2 showing the site in only one homolog. In contrast, the 5S rDNA sites on pairs 3 and 21 were found in interstitial positions on the long arm, while pair 10 exhibited a terminal position on the long arm. The 5S rDNA sites were syntenic with the heterochromatin regions, except for pairs 3, 10, and 21 (Fig. 1C).

In FISH experiments, the Rex-1 (Fig. 1D) and Rex-3 (Fig. 1E) elements displayed similar dispersion patterns, characterized by small clusters spread throughout the chromosomes in both euchromatic and heterochromatic regions. Notably, Rex-1 elements exhibited conspicuous clusters at the terminal positions of pairs 2, 3, 6, 8, 10, 21, 24 and 27, pairs 2 and 21 showing bitelomeric markings. In contrast, Rex-3 elements exhibited conspicuous clusters at the terminal positions of pairs 7, 9, and 17.

Discussion​

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The karyotype of G. inaequilabiatus analyzed in this study showed that the chromosome morphology, C-banding, and 5S FISH patterns differed from those described for the species in other cytogenetic studies performed in different collection sites. The unique chromosome morphology patterns found in G. inaequilabiatus containing 34m + 18sm + 2a obtained here were never observed in other specimens of the species (Tab. 1). This unique karyotype suggests that pericentric inversions (or other types of centromeric displacement) and/or deletions may be essential in the evolution and differentiation of the karyotype between populations of G. inaequilabiatus, given that its karyotypic structure is modified, but its diploid number of 2n = 54 chromosomes is still conserved. Additionally, the cytogenetic analysis of G. inaequilabiatus carried out by Fernandes-Matioli et al. (1998), which reported 2n = 52 chromosomes, contrasts with all previously and in this study analysed populations (Tab. 1). This discrepancy raises the possibility that what was identified as G. inaequilabiatus at that time may now be recognized as a distinct valid species.

TABLE 1 | Cytogenetic data from various populations of Gymnotus inaequilabiatus in the upper Paraná River basin. 2n = diploid number; m = metacentric; sm = submetacentric; st = subtelocentric; a = acrocentric; NORs = nucleolar organizer regions (number of bearing chromosomes utilizing of Ag-NOR and/or 18S rDNA-FISH); 5S = 5S rDNA cistrons number; I = interstitial marking. T = terminal marking. p = short arm.

Localities	2n	Karyotype structure	NORs	5S	References
Água da Madalena River, Botucatu, SP	54	42m+10sm+2a	1p (I)	34	Scacchetti et al. (2011)
Araquá River, Botucatu, SP	54	42m+10sm+2a	1p (I)	34	Scacchetti et al. (2011)
Campo Novo River, Bauru, SP	54	42m+10sm+2a	1p (I)	34	Scacchetti et al. (2011)
Mogi-Guaçu River, Pirassununga, SP	54	42m+10sm+2a	1p (I)	34	Scacchetti et al. (2011)
Claro River and Porto Primavera Dam, SP	52	40m+10sm+2st-a	23p (T)	–	Fernandes-Matioli et al. (1998)
Água do Ó Stream, Santa Fé, PR	54	34m+18sm+2a	1p (I)	31	Present study

Several other species share the same diploid chromosome number as G. inaequilabiatus, including G. paraguensis, G. cf. carapo, and G. mamiraua. These species primarily differ in their chromosome morphological patterns and the regions of repetitive DNA that can be identified through 5S rDNA analysis. This variation may indicate a recent speciation event (Silva, Margarido, 2005; Milhomem et al., 2012; Silva, 2015).

In Gymnotidae, considering the territorial behavior and its origin of dispersal through the Amazon basin (Albert, Crampton, 2003; Albert et al., 2005; Alves-Gomes, 2009), karyotypic plasticity can occur through structural and numerical chromosomal rearrangements due to its vicariant reproductive isolation. Vicariant reproductive isolation refers to the separation of populations due to geographical barriers, leading to genetic divergence. This, coupled with low migratory levels between individuals from different rivers, may also contribute to a more significant extension of repetitive DNA already observed in G. carapo (2n = 40/42), G. paraguensis (2n = 54), and G. inaequilabiatus (2n = 54), suggesting that it may be one of the main mechanisms of evolution in this group of fish (Fernandes-Matioli et al., 1998; Milhomem et al., 2008; Claro, Almeida-Toledo, 2010; Moysés et al., 2010).

The interstitial localization of NORs observed in the first pair of metacentrics following silver impregnation and FISH using an 18S rDNA probe is consistent across various populations of G. inaequilabiatus (Tab. 1) as well as related species such as G. pantherinus and G. cf. carapo. In G. inaequilabiatus, which has a chromosome number of 2n = 52 (Fernandes-Matioli et al., 1998), the presence of the NOR in a pair of submetacentric chromosomes reinforces the notion that rDNA sites exhibit significant dynamism. Simple NORs have been identified in several species within the Gymnotus genus, with the exception of G. jonasi, which has been found to possess NORs in three to five locations (Milhomem et al., 2011), and G. pantanal, where NORs appear in three chromosomes (Milhomem et al., 2007). Additionally, G. coatesi displays NORs located across 19 chromosomes following 18S-FISH analysis (Machado et al., 2017).

The heterochromatin patterns discussed in this article reveal a distribution of up to 27 pairs, predominantly located in centromeric and pericentromeric regions, with additional markings observed in the interstitial regions of some chromosomes. Other karyotypes analyzed in the two referenced articles exhibit similar heterochromatin distributions (Tab. 2); however, the number of markings varies, ranging from 26 (Scacchetti et al., 2011) to 54 (Margarido et al., 2007; Scacchetti et al., 2011; Silva et al., 2011; Sousa et al., 2017). The distribution of heterochromatin can differ among the studied Gymnotus species, with some species, such as G. carapo “Maranhão,” G. cf. pedanopterus, and G. paraguensis, displaying markings on nearly all chromosomes, while others, like G. sylvius, G. pantherinus, and G. cf. carapo, show fewer markings, with up to 14 pairs (Margarido et al., 2007; Scacchetti et al., 2011). Additionally, these regions may coincide with 5S rDNA, as seen in G. inaequilabiatus, G. paraguensis, G. carapo “Maranhão,” and G. mamiraua, or may not be fully coincident, as is the case for G. sylvius and G. pantherinus.

TABLE 2 | Cytogenetic data from different species in Gymnotus, considering the number of chromosome pairs carrying heterochromatic blocks and the number of chromosome pairs carrying 5S rDNA. Het = number of chromosomes carrying heterochromatin.

Species/cytotypes	Het	5S rDNA	References
G. bahianus	18	1	Almeida et al. (2015)
G. carapo “Maranhão”	17	12	Silva (2015)
G. inaequilabiatus	27	16	Present study
G. inaequilabiatus	27	17	Scachetti et al. (2011)
G. mamiraua	17	14	Silva et al. (2016)
G. pantanal	20	2	Sousa et al. (2017)
G. pantherinus	14	2	Scachetti et al. (2011)
G. paraguensis	27	19	Margarido et al. (2007); Silva et al. (2011)
G. paraguensis	27	17	Sousa et al. (2017)
G. sylvius	13	1	Scachetti et al. (2011)
G. sylvius	15	1	Sousa et al. (2017)

The 5S rDNA markers identified in this study display a distinct pattern across 16 chromosome pairs that harbor these sites. The presence of marking on only one homolog of chromosome pair 2 may be attributed to the association between heterochromatin and the 5S rDNA sites, which contributes to unequal crossing-over and the emergence of new rDNA loci. Alternatively, this pattern could be explained by the insertion of transposable elements into the 5S rDNA sequences of the other chromosome pairs, potentially leading to the dispersion of these sequences to the metacentric chromosome N^o 2 of G. inaequaliabiatus.

In a comparative study by Scacchetti et al. (2011), various specimens of G. inaequilabiatus collected from four different locations also exhibited numerous chromosomes carrying 5S rDNA, with counts reaching up to 17 pairs. In other species of Gymnotus, varying patterns of 5S rDNA distribution can be observed across different numbers of chromosome pairs. For instance, G. paraguensis shows 19 and 17 pairs (Silva et al., 2011; Sousa et al., 2017, respectively), while G. cf. carapo has 15 (Scacchetti et al., 2011), G. mamiraua has 14 (Silva et al., 2016), and G. carapo “Maranhão” displays 12 (Silva, 2015). In contrast, G. pantherinus and G. pantanal exhibit only 2 pairs (Scacchetti et al., 2011; Sousa et al., 2017), and G. bahianus and G. sylvius have just 1 each (Almeida et al., 2015; Scacchetti et al., 2011; Sousa et al., 2017). Additionally, studies conducted by Scacchetti et al. (2011, 2012) have documented different cytotypes of G. sylvius with 5S rDNA sites, which consistently display a single marked pair, albeit with variations in the marked pairs across different cytotypes.

The presence of numerous 5S rDNA sites in synteny with extensive blocks of heterochromatin may be associated with TEs. For example, mapping the Rex elements in the fish species demonstrated strong FISH signals in heterochromatin regions (Silva et al., 2020). TEs are DNA sequences capable of altering their position within a genome and are frequently located in heterochromatin regions, which are susceptible to chromosomal breaks. These breaks can result in the incorporation of these sequences into different chromosomes, thereby initiating an expansion of these sites. In this study, the synteny of 5S rDNA sites, heterochromatin, and Rex retroelements may have played a role in the dispersion of 5S rDNA sites across various chromosomes. In species such as G. sylvius and G. pantherinus, which exhibit small markings of heterochromatin, a corresponding low number of 5S rDNA sites can be observed (Tab. 2), indicating a reduced effectiveness of transposable elements. According to the report by Martins, Galetti Jr. (2001), two distinct classes of 5S rDNA containing non-transcribed spacers (NTS), located on different chromosomes in Leporinus, were demonstrated by Scacchetti et al. (2012) in a comparison between G. inaequilabiatus and G. sylvius. The G. inaequilabiatus with a more significant number of 5S rDNA markings exhibited these two classes on separate chromosomes, further contributing to a greater degree of dispersion of these sites. In contrast, the G. sylvius with fewer 5S rDNA markings displayed synteny between the two classes, resulting in a lower degree of dispersion of these sequences.

It is important to note that, to date, the examination of non-LTR retrotransposons in G. inaequilabiatus has not yet been conducted. The Rex-1 and Rex-3 retrotransposons discussed in this article exhibit a dispersed distribution throughout all chromosomes, maintaining synteny with regions of heterochromatin, euchromatin, and 5S rDNA. The association of Rex-1 and Rex-3 transposable elements with extensive blocks of heterochromatin may have significantly contributed to the proliferation of 5S rDNA regions across the chromosomes, given their highly dynamic nature. This phenomenon likely extends to other species within the genus that are reproductively isolated and possess substantial heterochromatin, serving to disperse 5S rDNA sequences throughout the genome.

According to Silva et al. (2016), a sequence resembling the Tc1/Mariner transposon has been identified within the genome of G. mamiraua, associated with a 5S rDNA fragment, which suggests the presence of a pseudogene. These sequences are also linked to heterochromatin blocks, potentially facilitating both the insertion and preservation of the pseudogenes. As a result, more than half of the chromosomes in this species of Gymnotus exhibit syntenic localization with both the 5S rDNA and the Tc1/Mariner transposon. Notably, 5S rDNA sites have been identified across 28 chromosomes.

Research on retroelements Rex-1, Rex-3, and Rex-6 has been conducted in various fish families, such as Loricariidae, Prochilodontidae, and Cichlidae. These studies aim to compare natural fish populations inhabiting different environments to determine whether various biotic and abiotic stresses can trigger the expression or transposition of mobile elements. Barbosa et al. (2014) reported a significant increase in the abundance of retroelements in populations residing in polluted regions compared to those in unpolluted areas. This finding suggests that environmental changes may stimulate the transcription of retrotransposons, as individuals must continuously adapt to survive in altered conditions. Additionally, small clusters of retroelements have been found in proximity to the 18S rDNA regions, contributing to the multiplicity of 18S rDNA sites observed in certain cichlid species (Gross et al., 2010; Schneider et al., 2013).

The present data are valuable in helping to understand karyotypic evolution in Gymnotus. Thus, pericentric inversions (or other types of centromeric displacement) and/or deletions seem essential in the evolution and differentiation of karyotypes among G. inaequilabiatus. Data on simple NORs and diploid number show that both characters are conserved in the species, as well as multiple 5S rDNA sites spread across several chromosomes, and that heterochromatin in synteny with retroelements may indicate an essential role in the dispersal of these ribosomal DNAs.

Acknowledgments​

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We thank Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), through the Dean of the Postgraduate and Research Department of the Universidade Estadual de Maringa (UEM-PPG), for the master’s scholarship granted to LPFG.

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Authors

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Lucas Pietro Ferrari Gianini¹, Ligia Carla Balini², Fernanda Errero Porto³, Luciana Andreia Borin-Carvalho² and Carlos Alexandre Fernandes^1,4

^[1]    Programa de Pós-Graduação em Biologia Comparada, Universidade Estadual de Maringá, Av. Colombo, 5790, 87020-900 Maringá, PR, Brazil. (LPFG) luquindia@gmail.com, (CAF) cafernandes@uem.br (corresponding author).

^[2]    Programa de Pós-Graduação em Genética e Melhoramento, Universidade Estadual de Maringá, Av. Colombo, 5790, 87020-900 Maringá, PR, Brazil. (LCB) ligia_balini@hotmail.com, (LABC) labcarvalho@uem.br.

^[3]    Departamento de Ciências Morfológicas, Universidade Estadual de Maringá, Av. Colombo, 5790, 87020-900 Maringá, PR, Brazil. (FEP) fepsaparolli@uem.br.

^[4]    Núcleo de Pesquisas em Limnologia, Ictiologia e Aquicultura (Nupelia), Universidade Estadual de Maringá, Av. Colombo, 5790, 87020-900 Maringá, PR, Brazil.

Authors’ Contribution

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Lucas Pietro Ferrari Gianini: Data curation, Formal analysis, Methodology, Writing-original draft, Writing-review and editing.

Ligia Carla Balini: Formal analysis, Methodology, Writing-original draft, Writing-review and editing.

Fernanda Errero Porto: Methodology, Writing-original draft, Writing-review and editing.

Luciana Andreia Borin-Carvalho: Formal analysis, Methodology, Writing-original draft, Writing-review and editing.

Carlos Alexandre Fernandes: Conceptualization, Data curation, Formal analysis, Methodology, Project administration, Supervision, Writing-original draft, Writing-review and editing.

Ethical Statement​

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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 Animal Experimentation Ethics Committee of the Universidade Estadual de Maringá (License Number: 6792170120 – CEUA/UEM). The experiments followed ethical conduct and, prior to euthanasia, the fish were anesthetized with an overdose of clove oil (Griffiths, 2000). The animals were captured with authorization from the Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio number 73763–2).

Competing Interests

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The author declares no competing interests.

How to cite this article

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Gianini LPF, Balini LC, Porto FE, Borin-Carvalho LA, Fernandes CA. Chromosome spreading of the 5S rDNA clusters in the karyotype of Gymnotus inaequilabiatus (Gymnotiformes: Gymnotidae): insights into the role of the Rex-1 and Rex-3 retrotransposable elements of this process. Neotrop Ichthyol. 2025; 23(1):e240089. https://doi.org/10.1590/1982-0224-2024-0089

Copyright​

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This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Distributed under

Creative Commons CC-BY 4.0

© 2025 The Authors.

Diversity and Distributions Published by SBI

Accepted December 18, 2024 by Izeni Farias

Submitted August 27, 2024

Epub March 14, 2025