© 2019. Published by The Company of Biologists Ltd | Biology Open (2019) 8, bio040352. doi:10.1242/bio.040352 RESEARCH ARTICLE Meiosis in the scorpion Tityus silvestris: new insights into achiasmatic chromosomes Bruno Rafael Ribeiro de Almeida, Renata Coelho Rodrigues Noronha, Marlyson Jeremias Rodrigues da Costa, Cleusa Yoshiko Nagamachi and Julio Cesar Pieczarka* ABSTRACT 1950a,b; Cunha and Pavan, 1954; Adilardi et al., 2016; Ojanguren- Achiasmatic male meiosis in scorpions is characterized by a high Affilastro et al., 2017). The presence of holocentric chromosomes frequency of gaps, asynaptic regions and multivalent associations. favors fixation of these rearrangements in several natural Here, we performed an immunocytogenetic analysis to investigate populations (Mattos et al., 2013). Direct consequences of such recombination, and synapsis and chromatin-remodeling events during changes are detected during prophase I events, as evidenced by meiosis of the scorpion Tityus silvestris. Our results demonstrate synaptic anomalies, such as gaps, asynapsed regions, loop-like that the synaptonemal complex (SC) begins its organization in the structures and a high frequency of heterosynapsis, in several zygotene stage and persists until metaphase I. The advancement of members of this genus (Schneider et al., 2009b). the synaptic process is related to the epigenetic modification histone Another interesting aspect of Tityus meiosis is the absence of H3 lysine 27 trimethylation (H3K27m3). The distribution and dynamics chiasma in males (Schneider et al., 2009a). Achiasmatic meiosis is patterns of variant γH2AX and recombinase Rad51 during achiasmatic not unique to this genus, occurring in all Scorpiones families, as meiosis suggests formation and repair of DNA double-strand well as in other organisms, such as plants, flatworms and insects breaks (DSBs) during early stages of prophase I. The epigenetic (Schneider et al., 2009a; Loidl, 2016). Chiasma formation is related modifications, histone H3 lysine 4 trimethylation (H3K4m3) and to the occurrence of DNA double-strand breaks (DSBs), created by histone H3 lysine 9 acetylation (H3K9ac), showed a dispersed the enzyme Spo11 (Moens et al., 2007). The recombinases, Rad51 distribution along the bivalents, suggesting that transcriptional (radiation sensitive 51) and DMC1 (disrupted meiotic cDNA1), activity is maintained constitutively during prophase I. However, perform reciprocal repair by subsequently associating with regions H3K9ac modifications are absent in constitutive heterochromatin of DSBs and connecting them to the homologous DNA strand, carrying the 45S rDNA in pachytene and post-pachytene stages. forming a heteroduplex (Gray and Cohen, 2016). The chiasma is the Collectively, our data demonstrate that T. silvestris exhibits adaptations cytological visualization of this process during the diplotene stage, to the achiasmatic mode, and suggest that epigenetic modifications after dissociation of the synaptonemal complex (SC). To date, no may act in the regulation of these mechanisms to favor the normal detailed studies of meiotic recombination in scorpions have been continuation of meiosis in this scorpion. reported, with the exception of an ultrastructural analysis of the SC of some Buthidae species that showed no recombination nodules KEY WORDS: Holocentric chromosome, Achiasmatic meiosis, (Shanahan and Hayman, 1990; Schneider et al., 2015). Scorpiones, Epigenetics, Synaptonemal complex Histones are protein components of nucleosomes that can undergo post-translational modifications through methylation, acetylation, INTRODUCTION ubiquitination, sumoylation or phosphorylation of different amino Tityus is the largest genus of the Buthidae family in the Neotropical acids present in the amino terminal tail (Venkatesh and Workman, region, comprising about 200 described species, some of which are 2015). Such changes reshape the chromatin, promoting the of great medical importance (Kovaríǩ et al., 2013; Lourenço, 2002, activation or inactivation of genes, or recruiting specific proteins to 2015). In recent years, these scorpions have been the subject of perform certain functions (Bannister and Kouzarides, 2011). During several meiotic studies, reflecting the fact that they exhibit extensive meiosis I in several organisms, insertions of histone variants in the multivalent associations during metaphase I. These meiotic chains chromatin are observed; these include the phosphorylated serine 136 may involve all chromosomes of the karyotype and are the result of histone variant H2AX (γH2AX) (Cabrero et al., 2007) and the occurrence of structural rearrangements of the fusion/fission methylation or acetylation of histone H3 at different amino acids type or reciprocal translocations in the heterozygous state (Piza, (Hayashi et al., 2005). Such changes may be related to the regulation of several meiotic processes, including recombination, condensation, segregation and transcriptional silencing of homologues (Moens Laboratório de Citogenética, Centro de Estudos Avançados em Biodiversidade, Instituto de Ciências Biológicas, Universidade Federal do Pará, Avenida Augusto et al., 2007; Naumova et al., 2013; Rahn et al., 2016). Corrêa, s/n, 66075-900, Guamá, Belém, Pará, Brazil. Thus, Tityus scorpions may be excellent models for epigenetic studies on achiasmatic meiotic chromosomes. In this study, we *Author for correspondence ([email protected]) performed a detailed analysis of the meiotic cycle of Tityus silvestris B.R.R.d.A., 0000-0002-6080-5826; R.C.R.N., 0000-0001-9150-8544; Pocock, 1988, with the objective of analyzing the synaptic behavior, M.J.R.d.C., 0000-0003-4415-2420; C.Y.N., 0000-0003-1516-2734; J.C.P., 0000- verifying the occurrence and repair of DSBs and investigating the 0003-2951-8877 relationship of these events to epigenetic modifications of bivalents This is an Open Access article distributed under the terms of the Creative Commons Attribution throughout prophase I of this species. In addition, information License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. about Tityus chromatin epigenetic dynamics may contribute to understanding the molecular organization of DNA in holocentric Received 17 November 2018; Accepted 15 April 2019 systems (Heckmann et al., 2013). Biology Open 1 RESEARCH ARTICLE Biology Open (2019) 8, bio040352. doi:10.1242/bio.040352 RESULTS Immunodetection of γH2AX demonstrated that the formation of Tityus silvestris has 2n=24 holocentric chromosomes, with one DSBs starts in pre-leptotene cells (Fig. 3A–D). In leptotene cells, large-size pair and 11 medium-size pairs (Fig. 1A). C-banding γH2AX signals were randomly distributed along the nucleus revealed only one heterochromatic block in the terminal region of (Fig. 3E–H). During the zygotene stage, γH2AX signals were co- pair 1 (Fig. 1B). During meiosis, post-pachytene cells exhibited located with SMC3 axes, polarized according to the bouquet bivalents, with homologues arranged parallel to each other, without configuration (Fig. 3I–L). In the early pachytene stage, γH2AX a configuration that evidences the presence of chiasma (Fig. 1C). signals increased in intensity, expanding along the synapsed SMC3 Fluorescence in situ hybridization (FISH) using 45S rDNA showed axes (Fig. 3M–P). In the intermediate pachytene stage, the number that this sequence is co-localized with the heterochromatin of pair 1 of γH2AX signals decreased drastically, with only a few visible (Fig. 1D). Telomeric sequences were observed only at chromosomal domains, most of which were restricted to the bivalent terminal ends (Fig. 1D). region (Fig. 3Q–T). No γH2AX signals were observed at the late Immunodetection of SMC3 (Structural Maintenance of pachytene stage (Fig. 3U–Z). Chromosomes 3) protein and FISH with a telomeric probe An analysis of the distribution of Rad51 revealed that foci of this showed that, in the leptotene stage, cohesin SMC3 was present in recombinase were first detected in the leptotene stage, located on the form of single filaments, dispersed along nuclei, or initiating the scattered SMC3 axes along the nucleus (Fig. 4A–D). In the bouquet formation (Fig. 2A–D). During the zygotene stage, SMC3 zygotene stage, Rad51 was observed only in the synapsed filaments and telomeres were polarized in the bouquet chromosome axes of the bouquet (Fig. 4E–H). During early and configuration, with synapsis initiation of homologs through intermediate phases of the pachytene stage, Rad5 foci were terminal regions (Fig. 2E–H). In early pachytene stage, the distributed along the SC, detectable on different regions of the bouquet configuration became disorganized, exhibiting longer synapsed axes or lateral to them (Fig. 4I–L). No Rad51 signals were double filaments, owing to advancement of the synapsis, that observed in the late pachytene stage (Fig. 4M–P). were continuous with single filaments (Fig. 2I–L). During the An analysis of the anti-H3K27m3 antibody-labeling pattern intermediate pachytene stage, bivalents were almost totally demonstrated that the proportion of histones bearing this synapsed, but with interstitial regions that had not yet completed modification increased in association with advancement of the the synapsis process (Fig. 2M–P); the synapsis of bivalents was synapsis. In the leptotene stage, H3K27m3 was located only at one completed in the late pachytene stage (Fig. 2Q–T). Post-pachytene pole