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Meiotic self-pairing of the Psalidodon (Characiformes, Characidae) iso-B chromosome: A successful perpetuation mechanism

Abstract

B chromosomes are non-essential additional genomic elements present in several animal and plant species. In fishes, species of the genus Psalidodon (Characiformes, Characidae) harbor great karyotype diversity, and multiple populations carry different types of non-essential B chromosomes. This study analyzed how the dispensable supernumerary B chromosome of Psalidodon paranae behaves during meiosis to overcome checkpoints and express its own meiosis-specific genes. We visualized the synaptonemal complexes of P. paranae individuals with zero, one, or two B chromosomes using immunodetection with anti-medaka SYCP3 antibody and fluorescence in situ hybridization with a (CA)15 microsatellite probe. Our results showed that B chromosomes self-pair in cells containing only one B chromosome. In cells with two identical B chromosomes, these elements remain as separate synaptonemal complexes or close self-paired elements in the nucleus territory. Overall, we reveal that B chromosomes can escape meiotic silencing of unsynapsed chromatin through a self-pairing process, allowing expression of their own genes to facilitate regular meiosis resulting in fertile individuals. This behavior, also seen in other congeneric species, might be related to their maintenance throughout the evolutionary history of Psalidodon.

Keywords:
Tetra fish; synaptonemal complex; SYCP3; meiotic silencing of unsynapsed chromatin (MSUC); fish meiosis

Introduction

Meiosis is a highly regulated process of reduced cell division occurring in germ cells, which allows for genetic recombination (Ma et al., 2014Ma H, Cooke HJ and Shi Q (2014) Meiosis: Recent progress and new opportunities. J Genet Genomics 41:83-85. ). During prophase I, homologous chromosomes are recognized and paired via synaptonemal complexes (SC), a protein structure formed between chromosomes. This structure is thought to mediate chromosome pairing, synapse, and even recombination. Meiotic silencing of unsynapsed chromatin (MSUC) significantly reduces transcriptional activity until the chromosomes reach their maximum state of pairing in the pachytene phase (Turner, 2015Turner J (2015) Meiotic silencing in mammals. Annu Rev Genet 49:395-412.). MSUC seems to be involved in controlling nonhomologous recombination (McKee and Handel, 1993McKee BD and Handel MA (1993) Sex chromosomes, recombination, and chromatin conformation. Chromosoma 102:71-80.; Zhang et al., 2014Zhang J, Zhang B, Su H, Birchler JA and Han F (2014) Molecular mechanisms of homologous chromosome pairing and segregation in plants. J Genet Genomics 41:117-123.) and activating the processes of cell death in cases of synapse failure (Faisal and Kauppi, 2016Faisal I and Kauppi L (2016) Sex chromosome recombination failure, apoptosis, and fertility in male mice. Chromosoma 125:227-235.).

B chromosomes are non-essential additional genomic elements that (in most cases) arise from autosomal chromosomes and follow their own evolutionary path through various types of genome rearrangements, losing the ability to properly pair and recombine with A chromosomes (Houben et al., 2014Houben A, Banaei-Moghaddam AM, Klemme S and Timmis JN (2014) Evolution and biology of supernumerary B chromosomes. Cell Mol Life Sci 71:467-478.). To perpetuate in the species, B chromosomes need to pair properly, avoiding meiosis disruption in a destructive manner. Cases where B chromosomes pair with each other to form bivalents, trivalents, and multivalents have been reported in literature (e.g. Pauls and Bertollo, 1983Pauls E and Bertollo LAC (1983) Evidence for a system of supernumerary chromosomes in Prochilodus scrofa steindachner, 1881 (Pisces, prochilodontidae). Caryologia 36:307-314.; Dias et al., 1998Dias AL, Foresti F and Oliveira C (1998) Synapsis in supernumerary chromosomes of Prochilodus lineatus (Teleostei: Prochilodontidae). Caryologia 51:105-113.; Basheva et al., 2010Basheva EA, Torgasheva AA, Sakaeva GR, Bidau C and Borodin PM (2010) A- and B-chromosome pairing and recombination in male meiosis of the silver fox (Vulpes vulpes L., 1758, Carnivora, Canidae). Chromosome Res 18:689-696.; Serrano et al., 2016Serrano EA, Araya-Jaime C, Suárez-Villota EY, Oliveira C and Foresti F (2016) Meiotic behavior and H3K4m distribution in B chromosomes of Characidium gomesi (Characiformes, Crenuchidae). Comp Cytogenet 10:255-268a.). This element frequently forms a univalent in cells with only one B chromosome, resulting in irregular meiosis. (Battaglia, 1964Battaglia E (1964) Cytogenetics of B-Chromosomes. Caryologia 17:245-299.; Pauls and Bertollo, 1983Pauls E and Bertollo LAC (1983) Evidence for a system of supernumerary chromosomes in Prochilodus scrofa steindachner, 1881 (Pisces, prochilodontidae). Caryologia 36:307-314.; Shi et al., 1988Shi L, Tang L, Ma K and Ma C (1988) Synaptonemal complex formation among supernumerary B chromosomes: An electron microscopic study on spermatocytes of Chinese raccoon dogs. Chromosoma 97:178-183.; Dias et al., 1998Dias AL, Foresti F and Oliveira C (1998) Synapsis in supernumerary chromosomes of Prochilodus lineatus (Teleostei: Prochilodontidae). Caryologia 51:105-113.; Basheva et al., 2010Basheva EA, Torgasheva AA, Sakaeva GR, Bidau C and Borodin PM (2010) A- and B-chromosome pairing and recombination in male meiosis of the silver fox (Vulpes vulpes L., 1758, Carnivora, Canidae). Chromosome Res 18:689-696.; Aquino et al., 2013Aquino CI, Abril VV and Duarte JMB (2013) Meiotic pairing of B chromosomes, multiple sexual system, and Robertsonian fusion in the red brocket deer Mazama americana (Mammalia, Cervidae). Genet Mol Res 12:3566-3574.; Pires et al., 2015Pires LB, Sampaio TR and Dias AL (2015) Mitotic and meiotic behavior of B chromosomes in Crenicichla lepidota: New report in the family cichlidae. J Hered 106:289-295.; Serrano et al., 2016Serrano EA, Araya-Jaime C, Suárez-Villota EY, Oliveira C and Foresti F (2016) Meiotic behavior and H3K4m distribution in B chromosomes of Characidium gomesi (Characiformes, Crenuchidae). Comp Cytogenet 10:255-268a.). Interestingly, iso-B chromosomes, reported in several species (Fletcher and Hewitt, 1988Fletcher HL and Hewitt GM (1988) Synaptonemal complexes of univalent B chromosomes in the grasshoppers Euthystira brachyptera and Myrmeleotettix maculatus. Heredity 60:383-386.; Dias et al., 1998Dias AL, Foresti F and Oliveira C (1998) Synapsis in supernumerary chromosomes of Prochilodus lineatus (Teleostei: Prochilodontidae). Caryologia 51:105-113.; Mestriner et al., 2000Mestriner CA, Galetti PM, Valentini SR, Ruiz IRG, Abel LDS, Moreira-Filho O and Camacho JPM (2000) Structural and functional evidence that a B chromosome in the characid fish Astyanax scabripinnis is an isochromosome. Heredity 85:1-9. ; Poletto et al., 2010Poletto AB, Ferreira IA and Martins C (2010) The B chromosomes of the African cichlid fish Haplochromis obliquidens harbour 18S rRNA gene copies. BMC Genet 11:1-8.; Valente et al., 2014Valente GT, Conte MA, Fantinatti BEA, Cabral-De-Mello DC, Carvalho RF, Vicari MR, Kocher TD and Martins C (2014) Origin and evolution of B chromosomes in the cichlid fish Astatotilapia latifasciata based on integrated genomic analyses. Mol Biol Evol 31:2061-2072.; Pires et al., 2015Pires LB, Sampaio TR and Dias AL (2015) Mitotic and meiotic behavior of B chromosomes in Crenicichla lepidota: New report in the family cichlidae. J Hered 106:289-295.), have the advantage of being composed of highly similar arms, allowing their perfect selfpairing, thus decreasing possible damage to cell division and increasing their chances of perpetuation (Battaglia, 1964Battaglia E (1964) Cytogenetics of B-Chromosomes. Caryologia 17:245-299.; Mestriner et al., 2000Mestriner CA, Galetti PM, Valentini SR, Ruiz IRG, Abel LDS, Moreira-Filho O and Camacho JPM (2000) Structural and functional evidence that a B chromosome in the characid fish Astyanax scabripinnis is an isochromosome. Heredity 85:1-9. ; Serrano-Freitas et al., 2020Serrano-Freitas EA, Silva DMZA, Ruiz-Ruano FJ, Utsunomia R, Araya-Jaime C, Oliveira C, Camacho JPM and Foresti F (2020) Satellite DNA content of B chromosomes in the characid fish Characidium gomesi supports their origin from sex chromosomes. Mol Genet Genom 295:195-207.).

The recently resurrected and expanded genus Psalidodon (Characiformes, Characidae) (Terán et al., 2020Terán GE, Benitez MF and Mirande JM (2020) Opening the Trojan horse: Phylogeny of Astyanax, two new genera and resurrection of Psalidodon (Teleostei: Characidae). Zool J Linn Soc 190:1217-1234.) includes species that formally belonged to the Astyanax genus, including P. paranae and P. scabripinnis, among others. These species harbor great karyotype diversity (Pazza et al., 2018Pazza R, Dergam JA and Kavalco KF (2018) Trends in karyotype evolution in Astyanax (Teleostei, Characiformes, Characidae): Insights from molecular data. Front Genet 9:131.) with several populations carrying different types of B chromosomes (Review in Silva et al., 2021Silva DMZA, Ruiz-Ruano FJ, Utsunomia R, Martín-Peciña M, Castro JP, Freire PP, Carvalho RF, Hashimoto DT, Suh A, Oliveira C et al. (2021) Long-term persistence of supernumerary B chromosomes in multiple species of Astyanax fish. BMC Biol 19:52. ). Among these species, the large metacentric B chromosome of P. scabripinnis was described as an isochromosome based on its self-pairing in meiotic cells and the symmetry of its arms as revealed by As51 satellite DNA mapping (Mestriner et al., 2000Mestriner CA, Galetti PM, Valentini SR, Ruiz IRG, Abel LDS, Moreira-Filho O and Camacho JPM (2000) Structural and functional evidence that a B chromosome in the characid fish Astyanax scabripinnis is an isochromosome. Heredity 85:1-9. ). This chromosome shares a common origin with the B chromosome of P. paranae (Silva et al., 2021Silva DMZA, Ruiz-Ruano FJ, Utsunomia R, Martín-Peciña M, Castro JP, Freire PP, Carvalho RF, Hashimoto DT, Suh A, Oliveira C et al. (2021) Long-term persistence of supernumerary B chromosomes in multiple species of Astyanax fish. BMC Biol 19:52. ), which is also an isochromosome based on the mapping of several repetitive DNAs (Silva et al., 2014Silva DMZA, Pansonato-Alves JC, Utsunomia R, Araya-Jaime C, Ruiz-Ruano FJ, Daniel SN, Hashimoto DT, Oliveira C, Camacho JPM, Porto-Foresti F et al. (2014) Delimiting the origin of a B chromosome by FISH mapping, chromosome painting and DNA sequence analysis in Astyanax paranae (Teleostei, Characiformes). PLoS ONE 9:33-36., 2016Silva DMZA, Daniel SN, Camacho JPM, Utsunomia R, Ruiz-Ruano FJ, Penitente M, Pansonato-Alves JC, Hashimoto DT, Oliveira C, Porto-Foresti F et al. (2016) Origin of B chromosomes in the genus Astyanax (Characiformes, Characidae) and the limits of chromosome painting. Mol Genet Genom 291:1407-1418., 2017Silva DMZA, Utsunomia R, Ruiz-Ruano FJ, Daniel SN, Porto-Foresti F, Hashimoto DT, Oliveira C, Camacho JPM and Foresti F. (2017) High-throughput analysis unveils a highly shared satellite DNA library among three species of fish genus Astyanax. Sci Rep. 7:1-12.). However, meiotic analyses have not yet been performed on this chromosome.

In this study, we analyzed the meiotic behavior of the B chromosome of P. paranae in individuals with one or two B chromosomes through synaptonemal complex immunodetection using an anti-medaka SYCP3 antibody and fluorescence in situ hybridization (FISH) using a (CA)15 microsatellite probe. We used an adaptation of the technique described by Araya-Jaime et al. (2015Araya-Jaime C, Serrano ÉA, Silva DMZA, Yamashita M, Iwai T, Oliveira C and Foresti F (2015) Surface-spreading technique of meiotic cells and immunodetection of synaptonemal complex proteins in teleostean fishes. Mol Cytogenet 8:4-9.) to unveil the pairing mechanism employed by these elements to overcome the meiosis checkpoints and achieve evolutionary success in Psalidodon. We also examined the upregulation of a meiosis-specific gene present on the B chromosome (mutS homolog 4 - msh4) in the gonads of B-carrying individuals, to determine the pairing behavior of these extra elements.

Material and Methods

Sampling and measurement of B chromosome number

Ten females, seven males and one intersex individual of P. paranae from the population of Cascatinha stream, Botucatu, São Paulo, Brazil (22° 53’ 22.5” S, 48° 29’ 22.4” W) were analyzed. The intersex individual was identified by the presence of oocytes and spermatozoa under a microscopy analysis of the gonads. Previous studies reported the presence of B chromosomes in some individuals of this population (Maistro et al., 1994bMaistro EL, Foresti F and Oliveira C (1994b) New occurrence of a macro B-chromosome in Astyanax scabripinnis paranae (Pisces, Characiformes, Characidae). Rev Bras Genet 17:153-156.; Porto-Foresti et al., 1997Porto-Foresti F, Oliveira C, Maistro EL and Foresti F (1997) Estimated frequency of B-chromosomes and population density of Astyanax scabripinnis paranae in a small stream. Braz J Genet 20:3.). The animals were collected in accordance with Brazilian environmental protection legislation (Collection Permission MMA/IBAMA/SISBIO-number 3245) and the procedures for fish sampling, maintenance, and analysis were performed in compliance with the Brazilian College of Animal Experimentation (COBEA) and approved (protocol 504) by the BIOSCIENCE INSTITUTE/UNESP ETHICS COMMITTEE ON THE USE OF ANIMALS (CEUA). The specimens were identified and deposited at the fish collection of the Laboratório de Biologia e Genética de Peixes, Botucatu, São Paulo, Brazil, under the voucher LBP19572 (P. paranae). The animals were anesthetized and dissected, and then mitotic chromosome preparations were obtained following the protocol described by Foresti et al. (1981Foresti F, Almeida-Toledo LF and Toledo-Filho SA (1981) Polymorphic nature of nucleolus organizer regions in fishes. Cytogenet. Cell Genet 31:137-144.). C-banding was performed according to the protocol described by Sumner (1972Sumner AT (1972) A simple technique for demonstrating centromeric heterochromatin. Exp Cell Res 75:304-306.) to improve accuracy in measuring the number of B chromosomes in the samples. Chromosome preparations were stained for 5 minutes with 5% Giemsa solution in phosphate buffer (pH= 6.7).

Synaptonemal complex analysis and fluorescence in situ hybridization

Synaptonemal complex preparations were performed according to Araya-Jaime et al. (2015Araya-Jaime C, Serrano ÉA, Silva DMZA, Yamashita M, Iwai T, Oliveira C and Foresti F (2015) Surface-spreading technique of meiotic cells and immunodetection of synaptonemal complex proteins in teleostean fishes. Mol Cytogenet 8:4-9.) using immature males and females. The SCs were stained using silver nitrate and immunodetected using an anti-medaka SYCP3 primary antibody/FITC-labeled secondary antibody. Subsequent FISH technique on mitotic preparations and meiotic spreads (SC-FISH) was conducted according to the protocol described by Cioffi et al. (2011Cioffi MB, Kejnovsky E and Bertollo LAC (2011) The chromosomal distribution of microsatellite repeats in the genome of the wolf fish Hoplias malabaricus, focusing on the sex chromosomes. Cytogenet Genome Res 132:289-296.) using the (CA)15 oligonucleotide as probe. This probe was directly labeled with Cy3 during synthesis, as described by Kubat et al. (2008Kubat Z, Hobza R, Vyskot B and Kejnovsky E (2008) Microsatellite accumulation on the Y chromosome in Silene latifolia. Genome 51:350-356.). This microsatellite was selected as a probe due to its high accumulation in the terminal region of both B chromosome arms at high visible sites (Silva et al., 2016Silva DMZA, Daniel SN, Camacho JPM, Utsunomia R, Ruiz-Ruano FJ, Penitente M, Pansonato-Alves JC, Hashimoto DT, Oliveira C, Porto-Foresti F et al. (2016) Origin of B chromosomes in the genus Astyanax (Characiformes, Characidae) and the limits of chromosome painting. Mol Genet Genom 291:1407-1418.) (Figure 1). Chromosomes were counterstained with 4’,6-Diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA). Images were captured with a digital camera (Olympus DP90) attached to an Olympus BX61 epifluorescence photomicroscope and acquired using CellSens Dimension (Olympus). Image treatment, optimization of brightness and contrast were performed using the Adobe Photoshop CS4 program. Meiotic phases were identified according to Toscani et al. (2008Toscani MA, Pigozzi MI, Bressa MJ, Papeschi AG (2008) Synapsis with and without recombination in the male meiosis of the leaf-footed bug Holhymenia rubiginosa (Coreidae, Heteroptera). Genetica 132:173-178.), Ribagorda et al. (2019Ribagorda M, Berríos S, Solano E, Ayarza E, Martín-Ruiz M, Gil-Fernández A, Parra MT, Viera A, Rufas JS, Capanna E et al. (2019) Meiotic behavior of a complex hexavalent in heterozygous mice for Robertsonian translocations: Insights for synapsis dynamics. Chromosoma 128:149-163.) and Spangenberg et al. (2019Spangenberg V, Arakelyan M, Galoyan E, Pankin M, Petrosyan R, Stepanyan I, Grishaeva T, Danielyan F and Kolomiets O (2019) Extraordinary centromeres: Differences in the meiotic chromosomes of two rock lizards species Darevskia portschinskii and Darevskia raddei. PeerJ 7:e6360).

Figure 1 -
Metaphase of P. paranae (2n=50+B) after FISH with a (CA)15 probe. Chromosomes were counterstained with DAPI. Bar = 10 µm.

Quantitative reverse transcription PCR (RT-qPCR)

Quantification of expression levels of msh4 gene was performed using gonads from females and males with and without B chromosomes. We analyzed four ovaries 0B, six ovaries 1B, four testes 0B and three testes 1B. After dissection, tissues were immediately frozen in liquid nitrogen and stored at -70 °C. RNA was extracted using the TRIzol® Kit (Invitrogen), following the manufacturer’s instructions. Subsequently, the samples were treated with DNAseI (Thermo Fisher Scientific) and were checked on 1% agarose gel and on a 2100 Bioanalyzer® (Agilent) equipment. Only RNA samples with an A260/280 ratio of 1.8-2.0, A260/230 ratio > 2.0, and RIN > 7 were used for subsequent analysis. The cDNA for each sample was synthesized using the High-Capacity cDNA Reverse Transcription Kit® (Thermo Fisher Scientific, USA) using 1 μg per sample of total RNA, following the manufacturer’s instructions. The cDNA obtained was diluted in RNase-DNase free water for a 1:50 working solution.

For RT-qPCR reactions, we used the primers for msh4 gene designed by Silva et al. (2021Silva DMZA, Ruiz-Ruano FJ, Utsunomia R, Martín-Peciña M, Castro JP, Freire PP, Carvalho RF, Hashimoto DT, Suh A, Oliveira C et al. (2021) Long-term persistence of supernumerary B chromosomes in multiple species of Astyanax fish. BMC Biol 19:52. ). Quantitative PCR was performed on QuantStudio™ 12K Flex Real-Time PCR Systems (Thermo Fisher Scientific, USA). The reactions were performed in a final volume of 10 µL, with 1 μl of cDNA, 5 μl of Power SYBR™ Green PCR Master Mix (Thermo Fisher Scientific) and 1 μl of each 5 µM primer. Cycle conditions were 95 °C for 10 min; 45 cycles of 95 °C for 15 s, and 60 °C for 1 min. Target and reference genes were analyzed simultaneously in duplicates for two independent samples. The normalized relative expression quantity (NREQ) was determined by the 2-∆∆Cq method (Livak and Schmittgen, 2001Livak KJ and Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods 25:402-408.). The msh4 expression levels were normalized using the hypoxanthine phosphoribosyltransferase 1 (hprt1) as the reference gene, with subsequent calibration to the average expression of the 0B group. The specificity of the PCR products was confirmed by dissociation curve analysis.

Two-group comparisons were performed by the Gardner-Altman estimation plot method devised by Ho et al. (2019Ho J, Tumkaya T, Aryal S, Choi H and Claridge-Chang A (2019) Moving beyond P values: Data analysis with estimation graphics. Nat Methods 16:565-566.) following Gardner and Altman’s design (1986Gardner MJ and Altman DG (1986) Confidence intervals rather than P values: Estimation rather than hypothesis testing. Br Med J 292:746-750. ), as implemented on https://www.estimationstats.com/.Estimation Statistics, https://www.estimationstats.com/.
https://www.estimationstats.com/...

Results

Meiocytes from five females (one 0B, three 1B and one not determined), five males (three 0B, one 1B and one 2B), and one intersex individual (0B) were analyzed. Overall, 48 meiocytes were analyzed, with an average of 4.8 cells analyzed per individual.

Silver nitrate staining of SCs revealed 25 bivalents in 0B individuals during the pachytene phase (Figure 2A), whereas in 1B individuals 26 completely paired SCs were observed (Figure 2B). The meiocytes of 2B individuals showed 26 SCs (Figure 2C). No differences between males and females were observed.

Figure 2 -
Detection of P. paranae synaptonemal complexes by silver nitrate staining (A, B, C). Diploid numbers of individuals are indicated in the lower left corner. Bar = 10 µm.

Immunodetection analysis of SC revealed 25 bivalents in the 0B individuals (Figure S1 Figure S1 - Detection of P. paranae synaptonemal complexes through immunodetection using anti-medaka SYCP3 antibody. ). The SC-FISH assay revealed 25 fully synaptized bivalents in 1B individuals. As the B chromosome has conspicuous (CA)15 sites in both terminal regions (Figure 1), the assay also showed a SC marked by the (CA)15 probe in only one of the ends, corresponding to the self-paired B chromosome (Figure 3 A-C). In addition to 25 fully synaptized SCs, SC-FISH in 2B individual preparations showed a SC association marked on only one end by the (CA)15 probe, corresponding to two self-paired and close B chromosomes (Figure 3 E-G). A region not fully associated with the two SCs was observed in this structure (Figure 3E). In some pachytene preparations of 2B individuals, 25 normal bivalents plus two SCs marked by the (CA)15 probe were seen at only one of the ends, corresponding to two independently self-paired B chromosomes (Figure 3 I-K).

Figure 3 -
Detection of P. paranae synaptonemal complexes through immunodetection using an anti-medaka SYCP3 antibody (A, E, I) and FISH with a (CA)15 probe (B, F, J). Diploid number of individuals are indicated in the lower left corner (A, E, I). In the inset of e, note a region not fully associated in the lower half of the SC association formed by two self-paired B chromosomes. C, G, and K show the combination of images obtained by immunodetection and FISH. D, H, and L present an illustration of the meiotic behavior of the B chromosome. In all individuals, B chromosomes show a self-pairing behavior and in 2B individuals, they can stay close or separated. In the first case, they can appear not perfectly paired to each other. Bar = 10 µm.

The meiotic behavior of two self-paired B chromosomes can be observed in Figure 4. In early diplotene, the formation of a “ring-like” figure can be observed with the chromosomes united at their ends (Figure 4 A-C). In more advanced stages of diplotene, complete dissociation of the B chromosomes shown as univalent was observed, apparently in a state of dissociation more advanced in relation to the chromosomes of the A complement (Figure 4D). The pairing patterns of P. paranae B chromosomes are illustrated in Figure 3 D, H, L.

Figure 4 -
Diplotene images of P. paranae after immunodetection using anti-medaka SYCP3 antibody and SC-FISH assay using the (CA)15 probe (A, B, C) and the late-diplotene stages after immunodetection using the anti-medaka SYCP3 antibody (D). Diploid numbers of individuals are indicated in the lower left corners of images a and d. Note that in d, the B chromosomes are entirely dissociated, and are shown as univalents. Bar = 10 µm.

No variation was observed in the number of B chromosomes among the meiotic cells of each specimen. Thus, the 0B, 1B, and 2B individuals presented zero, one, and two B chromosomes in all their meiotic cells, respectively. No regions with failure in synapsis were observed among the chromosomes of the A complement in individuals of both sexes of P. paranae (Figures 2 and 3), suggesting the absence of proto-sex chromosomes at differentiation status (Murata et al., 2016Murata C, Sawaya H, Nakata K, Yamada F, Imoto I and Kuroiwa A (2016) The cryptic Y-autosome translocation in the small Indian mongoose, Herpestes auropunctatus, revealed by molecular cytogenetic approaches. Chromosoma 125:807-815.). To the best of our knowledge, no sex-related chromosomal polymorphism has been described in the genus Psalidodon.

Transcription levels for the msh4 gene were about the 12-fold effect size (mean differences) in 1B ovaries compared to those in 0B ones. In the testes, this difference was about 4-fold (Figure 5, Table S1 Table S1 - Relative transcription levels for the gene msh4 in B-carrying and B-lacking individuals. ). These results demonstrate upregulation of the msh4 gene in the ovaries and testes of B-carrying individuals.

Figure 5 -
Gardner-Altman estimation plots showing msh4 transcription levels in 0B and 1B individuals, as analyzed by RT-qPCR. Both groups are plotted on the left axes, and the mean difference (effect size) is plotted on a floating axe at the right as a bootstrap sampling distribution. The mean difference is depicted as a black dot, and the 95% confidence interval is indicated by the ends of the vertical error bar. NREQ = normalized relative expression quantity.

Discussion

Meiotic self-pairing of iso-B chromosomes can allow them to advance through meiosis checkpoints and may be responsible for the success of their perpetuation (review in Clark et al., 2017Clark FE, Conte MA, Ferreira-Bravo IA, Poletto AB, Martins C and Kocher TD (2017) Dynamic sequence evolution of a sex-Associated b chromosome in lake Malawi cichlid fish. J Hered 108:53-62.; Silva et al., 2021Silva DMZA, Ruiz-Ruano FJ, Utsunomia R, Martín-Peciña M, Castro JP, Freire PP, Carvalho RF, Hashimoto DT, Suh A, Oliveira C et al. (2021) Long-term persistence of supernumerary B chromosomes in multiple species of Astyanax fish. BMC Biol 19:52. ). This behavior could allow B chromosomes without homologous pairs to circumvent the MSUC process. Thus, a selection pressure favoring the B iso-chromosomes would exist, as the MSUC process might lead to cell death in cases of synapse failure (Faisal and Kauppi, 2016Faisal I and Kauppi L (2016) Sex chromosome recombination failure, apoptosis, and fertility in male mice. Chromosoma 125:227-235.). Our results thus demonstrate that B chromosomes of P. paranae, previously described as isochromosomes (Silva et al., 2014Silva DMZA, Pansonato-Alves JC, Utsunomia R, Araya-Jaime C, Ruiz-Ruano FJ, Daniel SN, Hashimoto DT, Oliveira C, Camacho JPM, Porto-Foresti F et al. (2014) Delimiting the origin of a B chromosome by FISH mapping, chromosome painting and DNA sequence analysis in Astyanax paranae (Teleostei, Characiformes). PLoS ONE 9:33-36.), can self-pair as observed in the B chromosome of its sister species, P. scabripinnis (Mestriner et al., 2000Mestriner CA, Galetti PM, Valentini SR, Ruiz IRG, Abel LDS, Moreira-Filho O and Camacho JPM (2000) Structural and functional evidence that a B chromosome in the characid fish Astyanax scabripinnis is an isochromosome. Heredity 85:1-9. ). After diverging for millions of years (Silva et al., 2021Silva DMZA, Ruiz-Ruano FJ, Utsunomia R, Martín-Peciña M, Castro JP, Freire PP, Carvalho RF, Hashimoto DT, Suh A, Oliveira C et al. (2021) Long-term persistence of supernumerary B chromosomes in multiple species of Astyanax fish. BMC Biol 19:52. ), both chromosomes still have self-pairing capacity, reinforcing the role of this behavior as one of the main mechanisms responsible for maintaining such chromosomes in the populations of Psalidodon.

The success of B chromosomes is mainly related to their accumulation in populations due to transmission rates higher than 0.5, as expected by Mendelian laws. This process is called drive and occurs due to the patterns of B chromosome segregation (Camacho et al., 2005Camacho JPM (2005) B Chromosomes. In: Gregory TR (ed). The evolution of the genome. Elsevier, San Diego, pp. 223-286.). As the techniques used here did not enable us to observe all the meiotic phases, we cannot verify whether the patterns of B chromosome segregation are influenced by other factors (Wu et al., 2019Wu D, Ruban A, Fuchs J, Macas J, Novák P, Vaio M, Zhou Y and Houben A (2019) Nondisjunction and unequal spindle organization accompany the drive of Aegilops speltoides B chromosomes. New Phytol 223:1340-1352.). The increased frequency of B chromosomes in the analyzed population (Goes, in prep.) might be related to their ability to circumvent the MSUC process and remain undetected or cause deleterious effects in carriers.

B chromosomes are mainly composed of repetitive DNA sequences related to chromosome folding, recombination, meiotic pairing, and even disjunction in the germ line tissue (review in Garrido-Ramos, 2017Garrido-Ramos MA (2017) Satellite DNA: An evolving topic. Genes (Basel) 8:230.); further, in several cases, pairing between B chromosomes was attributed to their homologous content (Pauls and Bertollo, 1983Pauls E and Bertollo LAC (1983) Evidence for a system of supernumerary chromosomes in Prochilodus scrofa steindachner, 1881 (Pisces, prochilodontidae). Caryologia 36:307-314.; Switonski et al., 1987Switonski M, Gustavsson I, Höjer K and Plöen L (1987) Synaptonemal complex analysis of the B-chromosomes in spermatocytes of the silver fox (Vulpes vulpes Desm.). Cytogenet Cell Genet 45:84-92.; Kolomiets et al., 1988Kolomiets OL, Borbiev TE, Safronova LD, Borisov YM and Bogdanov YF (1988) Synaptonemal analysis of B chromosome behaviour in meiotic prophase I in the East-Asiatic mouse Apodemus peninsulae (Muridae, Rodentia). Cytogenet Cell Genet 48:183-187.; Maistro et al., 1994aMaistro EL, Dias AL, Foresti F, Oliveira C and Moreira-Filho O (1994a) Natural triploidy in Astyanax scabripinnis (Pisces, Characidae) and simultaneous occurrence of macro B-chromosomes. Caryologia 47:233-239.; De Brito Portela-Castro et al., 2001Portela-Castro ALB, Julio Júnior HF and Nishiyama PB (2001) New occurrence of microchromosomes B in Moenkhausia sanctaefilomenae (Pisces, Characidae) from the Paraná River of Brazil: Analysis of the synaptonemal complex. Genetica 110:277-283.; Serrano et al., 2016Serrano EA, Araya-Jaime C, Suárez-Villota EY, Oliveira C and Foresti F (2016) Meiotic behavior and H3K4m distribution in B chromosomes of Characidium gomesi (Characiformes, Crenuchidae). Comp Cytogenet 10:255-268a.). In this manner, repetitive DNA sequences symmetrically distributed on B chromosomes of P. paranae and P. scabripinnis (Mestriner et al., 2000Mestriner CA, Galetti PM, Valentini SR, Ruiz IRG, Abel LDS, Moreira-Filho O and Camacho JPM (2000) Structural and functional evidence that a B chromosome in the characid fish Astyanax scabripinnis is an isochromosome. Heredity 85:1-9. ; Silva et al., 2014Silva DMZA, Pansonato-Alves JC, Utsunomia R, Araya-Jaime C, Ruiz-Ruano FJ, Daniel SN, Hashimoto DT, Oliveira C, Camacho JPM, Porto-Foresti F et al. (2014) Delimiting the origin of a B chromosome by FISH mapping, chromosome painting and DNA sequence analysis in Astyanax paranae (Teleostei, Characiformes). PLoS ONE 9:33-36., 2016Silva DMZA, Daniel SN, Camacho JPM, Utsunomia R, Ruiz-Ruano FJ, Penitente M, Pansonato-Alves JC, Hashimoto DT, Oliveira C, Porto-Foresti F et al. (2016) Origin of B chromosomes in the genus Astyanax (Characiformes, Characidae) and the limits of chromosome painting. Mol Genet Genom 291:1407-1418., 2017Silva DMZA, Utsunomia R, Ruiz-Ruano FJ, Daniel SN, Porto-Foresti F, Hashimoto DT, Oliveira C, Camacho JPM and Foresti F. (2017) High-throughput analysis unveils a highly shared satellite DNA library among three species of fish genus Astyanax. Sci Rep. 7:1-12.; Artoni et al., 2015Artoni RF, Castro JP, Jacobina UP, Lima-Filho PA, Costa GWWF and Molina WF (2015) Inferring diversity and evolution in fish by means of integrative molecular cytogenetics. Sci World J 2015:365787) would then be responsible for self-pairing and symmetry maintenance could be essential for perpetuating this advantageous behavior.

In individuals with two B chromosomes, these elements are self-paired, even though the association between them is facultative (Figure 3 G, K). As discussed, self-pairing of B chromosomes may represent an adaptive advantage. Association of two self-paired B chromosomes might not be required to circumvent the MSUC process, because selfpaired B chromosomes do not have unsynapsed regions. The association between two B chromosomes observed here appears to result from their homologous content recognition and/or their physical proximity to specific territories inside the nucleus (Schemczssen-Graeff et al., 2020Schemczssen-Graeff Z, Barbosa P, Castro JP, Silva DMZA, Almeida MC, Moreira-Filho O and Artoni RF (2020) Dynamics of replication and nuclear localization of the B Chromosome in kidney tissue cells in Astyanax scabripinnis (Teleostei: Characidae). Zebrafish 17:147-152.). When B chromosomes are associated, we observe synaptic regions forming between chromatids (Figure 4A), as the employed technique preserves chromosomal associations and avoids the “squash” procedure (Araya-Jaime et al., 2015Araya-Jaime C, Serrano ÉA, Silva DMZA, Yamashita M, Iwai T, Oliveira C and Foresti F (2015) Surface-spreading technique of meiotic cells and immunodetection of synaptonemal complex proteins in teleostean fishes. Mol Cytogenet 8:4-9.).

Self-pairing of B chromosomes can favor homogenization of their sequences through regular recombination (Rice, 1994Rice WC (1994) Degeneration of a nonrecombining chromosome. Science 263:230-232.; Basheva et al., 2010Basheva EA, Torgasheva AA, Sakaeva GR, Bidau C and Borodin PM (2010) A- and B-chromosome pairing and recombination in male meiosis of the silver fox (Vulpes vulpes L., 1758, Carnivora, Canidae). Chromosome Res 18:689-696.). The low diversification of ITS1 and ITS2 regions on the B chromosome of P. paranae was attributed to this phenomenon by Silva et al. (2014Silva DMZA, Pansonato-Alves JC, Utsunomia R, Araya-Jaime C, Ruiz-Ruano FJ, Daniel SN, Hashimoto DT, Oliveira C, Camacho JPM, Porto-Foresti F et al. (2014) Delimiting the origin of a B chromosome by FISH mapping, chromosome painting and DNA sequence analysis in Astyanax paranae (Teleostei, Characiformes). PLoS ONE 9:33-36.). Regular recombination can also be responsible for the conservation of protein-coding sequences observed on the B chromosomes of P. paranae and P. scabripinnis (Silva et al., 2021Silva DMZA, Ruiz-Ruano FJ, Utsunomia R, Martín-Peciña M, Castro JP, Freire PP, Carvalho RF, Hashimoto DT, Suh A, Oliveira C et al. (2021) Long-term persistence of supernumerary B chromosomes in multiple species of Astyanax fish. BMC Biol 19:52. ). This conservation was indicated by the presence of a few B-specific SNPs and complete coding regions on these chromosomes (Silva et al., 2021Silva DMZA, Ruiz-Ruano FJ, Utsunomia R, Martín-Peciña M, Castro JP, Freire PP, Carvalho RF, Hashimoto DT, Suh A, Oliveira C et al. (2021) Long-term persistence of supernumerary B chromosomes in multiple species of Astyanax fish. BMC Biol 19:52. ). Similarly, natural selection has preserved the sequences of apparently intact and functional c-KIT on the B chromosome of foxes (Graphodatsky et al., 2005Graphodatsky AS, Kukekova AV, Yudkin DV, Trifonov VA, Vorobieva NV, Beklemisheva VR, Perelman PL, Graphodatskaya DA, Trut LN, Yang F et al. (2005) The proto-oncogene C-KIT maps to canid B-chromosomes. Chromosome Res 13:113-122.; Yudkin et al., 2007Yudkin D, Trifonov V, Kukekova A, Vorobieva N, Rubtsova N, Yang FD, Acland GMC, Ferguson-Smith MAB and Graphodatsky ASA (2007) Mapping of KIT adjacent sequences on canid autosomes and B chromosomes. Cytogenet. Genome Res 116:100-103.). In this case, recombination of B chromosomes was considered a protective mechanism for copies of this gene against mutational meltdown and degeneration (Basheva et al., 2010Basheva EA, Torgasheva AA, Sakaeva GR, Bidau C and Borodin PM (2010) A- and B-chromosome pairing and recombination in male meiosis of the silver fox (Vulpes vulpes L., 1758, Carnivora, Canidae). Chromosome Res 18:689-696.).

Unpaired B chromosomes can be silenced at meiosis (Serrano et al., 2016Serrano EA, Araya-Jaime C, Suárez-Villota EY, Oliveira C and Foresti F (2016) Meiotic behavior and H3K4m distribution in B chromosomes of Characidium gomesi (Characiformes, Crenuchidae). Comp Cytogenet 10:255-268a.), as observed in unpaired regions in the fungus Neurospora crassa (Sordariales, Sordariaceae) (Shiu and Metzenberg, 2002Shiu PKT and Metzenberg RL (2002) Meiotic silencing by unpaired DNA: Properties, regulation and suppression. Genetics 161:1483-1495.) and in mice (Turner et al., 2005Turner JMA, Mahadevaiah SK, Fernandez-Capetillo O, Nussenzweig A, Xu X, Deng CX, Burgoyne PS (2005) Silencing of unsynapsed meiotic chromosomes in the mouse. Nat Genet 37:41-47.). Self-pairing of B chromosomes, observed in P. paranae (this study) and in P. scabripinnis by Mestriner et al. (2000Mestriner CA, Galetti PM, Valentini SR, Ruiz IRG, Abel LDS, Moreira-Filho O and Camacho JPM (2000) Structural and functional evidence that a B chromosome in the characid fish Astyanax scabripinnis is an isochromosome. Heredity 85:1-9. ), could allow them to escape from silencing epigenetic modifications of the MSUC process, resulting in the transcriptional activity of these chromosomes (Serrano et al., 2016Serrano EA, Araya-Jaime C, Suárez-Villota EY, Oliveira C and Foresti F (2016) Meiotic behavior and H3K4m distribution in B chromosomes of Characidium gomesi (Characiformes, Crenuchidae). Comp Cytogenet 10:255-268a.). Accordingly, Silva et al. (2021Silva DMZA, Ruiz-Ruano FJ, Utsunomia R, Martín-Peciña M, Castro JP, Freire PP, Carvalho RF, Hashimoto DT, Suh A, Oliveira C et al. (2021) Long-term persistence of supernumerary B chromosomes in multiple species of Astyanax fish. BMC Biol 19:52. ) identified the expression of B-specific transcripts of important genes related to the cell cycle and gonadal development in the ovaries of both species, revealing that the B chromosomes are not silenced during meiosis.

Among them, the msh4 gene, a meiosis-specific homologue of the bacterial MutS protein, is essential for meiotic recombination and proper segregation of homologous chromosomes. In mice, meiosis is severely disrupted in MSH4 mutant males and females, resulting in infertility (Kneitz et al., 2000Kneitz B, Cohen PE, Avdievich E, Zhu L, Kane MF, Hou H, Kolodner RD, Kucherlapati R, Pollard JW and Edelmann W (2000) MutS homolog 4 localization to meiotic chromosomes is required for chromosome pairing during meiosis in male and female mice. Genes Dev 14:1085-1097.; Dai et al., 2017Dai J, Voloshin O, Potapova S and Camerini-Otero RD (2017) Meiotic knockdown and complementation reveals essential role of RAD51 in mouse spermatogenesis. Cell Rep 18:1383-1394.). We consider that transcription of the msh4 gene on the B chromosomes of P. paranae and P. scabripinnis helps ensure the required conditions for the proper division of cells carrying extra elements with atypical pairing. We observed upregulation of this gene in the ovaries and testes of 1B individuals of P. paranae, highlighting their role during the divisions leading to gonad formation in both sexes.

In conclusion, this study shows that the B chromosomes of P. paranae and P. scabripinnis can express their own meiosis-specific genes by escaping the MSUC through meiotic self-pairing. This ancient behavior results in regular meiosis and fertile individuals, allowing the evolutionary success and maintenance of B chromosomes throughout the evolutionary history of Psalidodon. Extension of these analyses beyond the iso-B chromosomes of Psalidodon is necessary to comprehend how non-isochromosome behavior perpetuates in this genus. The use of controlled crosses could provide information about the segregation patterns and transmission rates of the B chromosome in P. paranae.

Acknowledgements

This study was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) (grants 2014/09634-5, 2017/15608-5 to FF; 2013/24143-5, 2017/22447-8 to DMZAS and 2018/20610-1, 2016/09204-6, 2014/26508-3 to CO), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (grant 306054/2006-0 to CO), Doctorado en el Exterior, Becas-Chile (grant 72110694 to CAJ). We are thankful to Maria Soledad Berrios for providing helpful suggestions.

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Internet Resources

Edited by

Associate Editor: Vera Maria Fonseca de Almeida e Val

Publication Dates

  • Publication in this collection
    04 Oct 2021
  • Date of issue
    2021

History

  • Received
    20 Mar 2021
  • Accepted
    14 Aug 2021
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