In this study, we investigated the mitotic and meiotic chromosomes of 11 Buthidae scorpion species, belonging to three genera (Ananteris, Rhopalurus and Tityus), to obtain detailed knowledge regarding the mechanisms underlying the intraspecific and/or interspecific diversity of chromosome number and the origin of the complex chromosome associations observed during meiosis. The chromosomes of all species did not exhibit a localised centromere region and presented synaptic and achiasmatic behaviour during meiosis I. Spermatogonial and/or oogonial metaphase cells of these buthids showed diploid numbers range from 2n = 6 to 2n = 28. In most species, multivalent chromosome associations were observed in pachytene and postpachytene nuclei. Moreover, intraspecific variability associated with the presence or absence of chromosome chains and the number of chromosomes in the complex meiotic configurations was observed in some species of these three genera. Silver-impregnated cells revealed that the number and location of nucleolar organiser regions (NORs) remained unchanged despite extensive chromosome variation; notably, two NORs located on the terminal or subterminal chromosome regions were commonly observed for all species. C-banded and fluorochrome-stained cells showed that species with conspicuous blocks of heterochromatin exhibited the lowest rate of chromosomal rearrangement. Based on the investigation of mitotic and meiotic cells, we determined that the intraspecific variability occurred as a consequence of fission/fusion-type chromosomal rearrangements in Ananteris and Tityus species and reciprocal translocation in Rhopalurus species. Furthermore, we verified that individuals presenting the same diploid number differ in structural chromosome organisation, giving rise to intraspecific differences of chromosome association in meiotic cells (bivalent-like elements or chromosome chains).

The present study elevates the number of cytogenetically analyzed ctenid species and genera from two to eight and six, respectively, presenting comparisons between chromosomal data obtained and the phylogenetic hypothesis proposed in the literature. Six ctenid species presented 13 autosomal pairs, exhibiting either X1X2O (Ctenus ornatus, Ctenus sp., Parabatinga brevipes and Phoneutria nigriventer) or X1X2X3O sex chromosome systems (Nothroctenus sp. and Viracucha andicola). Asthenoctenus borellii showed 2n ♂ = 20 + X1X2O. In all species, the chromosomes were telocentric. Some cells of one C. ornatus specimen exhibited one extra chromosome that, considering the behavioral similarities between the two chromosomes, can be considered to be supernumerary, derived from or giving rise to a sex chromosome. Silver impregnation revealed nucleolar organizer regions on one autosomal pair of C. ornatus and P. nigriventer (Cteninae) and two pairs of V. andicola (Acanthocteninae). Chromosomal data suggests that the X1X2X3O system arose several times in the evolution of entelegyne spiders, and that conversion of an X1X2O system into an X1X2X3O system and vice-versa has been a relatively common event in spiders. All the chromosomal data corroborate the close relationship between Ctenus and Phoneutria, the placement of P. brevipes within Cteninae, the placement of Anahita in a separated branch within Cteninae, and the inclusion of A. borellii in a distinct group within the ctenids (Viridasiinae), all of which are as proposed by phylogenetic hypotheses available in the literature.

Buthid scorpions exhibit a high variability in diploid number within genera and even within species. Cytogenetically, Buthidae differs from other families of Scorpiones based on its low diploid numbers, holocentric chromosomes, and complex chromosomal chains, which form during meiosis. In this study, we analyzed the distribution of the 45S ribosomal DNA (rDNA) genes in the mitotic and meiotic chromosomes of seven buthid species belonging to the genera Rhopalurus and Tityus with the ultimate goal of elucidating the chromosome organization in these scorpions. The chromosome number ranged from 2n=6 to 2n=28. Despite the high variance in diploid number, all species examined carried their 45S rDNA sites in the terminal region of exactly two chromosomes. Analyses of meiotic cells revealed 45S rDNA clusters in the chromosomal chains of Rhopalurus agamemnon, Tityus bahiensis, Tityus confluens, and Tityus martinpaechi, or in bivalent-like configuration in Rhopalurus rochai, Tityus bahiensis, Tityus confluens, Tityus fasciolatus, and Tityus paraguayensis. In the species examined, the 45S rDNA sites colocalized with constitutive heterochromatin regions. In light of the high chromosome variability and maintenance of number and terminal position of 45S rDNA sites in buthids, the heterochromatin may act to conserve the integrity of the ribosomal genes.

Testicular cells of 4 buthid scorpions, Rhopalurus agamemnon (2n = 28), R. rochai (2n = 28), Tityus bahiensis (2n = 6), and T. fasciolatus (2n = 14), which show different types of chromosomal configurations in meiosis I, were subjected to cellular microspreading in order to (1) obtain knowledge about the organization and behavior of the synaptonemal complex (SC), and (2) acquire data about the mechanisms responsible for inter- and intraindividual chromosomal variation within Buthidae. Ultrastructural analysis of microspread nuclei revealed SCs with a well-preserved structure until late substages of prophase I, but did not detect kinetochore plates and recombination nodules. Pachytene cells of R. agamemnon, R. rochai and T. bahiensis exhibited single and unsynapsed axes continuous with totally synapsed SCs, indicating the occurrence of heterozygous chromosomal rearrangements. Although chromosome chains were not observed in T. fasciolatus, the presence of gaps and interlocks points out that this species also carries heterozygous rearrangements, involving a small chromosome segment. Especially in R. rochai, the cellular microspreading analysis was useful to clarify the origin of inter- and intraindividual variation in the number of bivalent-like elements and in the number of chromosomes involved in multivalent associations. It was found that more chromosomes were involved in rearrangements than previously established through investigations using light microscopy alone.

Scorpions represent an intriguing group of animals characterized by a high incidence of heterozygous chromosomal rearrangements. In this work, we examined six species of Tityus (Archaeotityus) from Brazilian fauna with a particular focus on elucidating the rearrangements responsible for the intraspecific variability of diploid number and the presence of long chromosomal chains in meiosis. To access any interpopulation diversity, we also studied individuals from four species representing distinct localities. Most species demonstrated intraspecific polymorphism in diploid number (2n = 19 and 2n = 20 in T. clathratus, T. mattogrossensis, and T. pusillus, 2n = 16, 2n = 17 and 2n = 18 in T. paraguayensis, and 2n = 16 and 2n = 24 in T. silvestris) and multi-chromosomal associations during meiosis I, which differed even among individuals with the same chromosome number. In some species, the heterozygous rearrangements were not fixed, resulting such as in Tityus clathatrus, in 11 different chromosomal configurations recognized within a same population. Based on meiotic chromosome behaviour, we suggested that independent rearrangements (fusion/fission and reciprocal translocations), occurring in different combinations, originated the multi-chromosomal chains. To evaluate the effects of these chromosome chains on meiotic segregation, we applied the chi-square test in metaphase II cells. The non-significant occurrence of aneuploid nuclei indicated that non-disjunction was negligible in specimens bearing heterozygous rearrangements. Finally, based on our analysis of many chromosome characteristics, e.g., holocentricity, achiasmate meiosis, endopolyploidy, ability to segregate heterosynaptic or unsynapsed chromosomes, ()n sequence located in terminal regions of rearranged chromosomes, we suggest that the maintenance of multi-chromosomal associations may be evolutionarily advantageous for these species.

No abstract

The family Araneidae is the third largest among spiders and the third most studied from a cytogenetical point of view. In spite of this, only 2% of all araneids have been karyotyped. The majority of araneids analyzed possess 2n = 24 chromosomes in males; however, the study of additional species could reveal unusual karyotype characteristics. Thus, the aim of this work is to analyze chromosomally, for the first time, six species belonging to three araneid genera from Brazil. The specimens of Alpaida leucogramnui (White 1841), Alpaida tnmeata (Keyserling 1865), Alpaida veniliae (Keyserling 1865), Parawixia kochi (Taezanowski 1873), ParawLxki velutina (Taezanowski 1878) and Wagneriana sp. were collected in Barque Nacional de Ilha Grande and in the municipality of Rio Claro. The gonads were treated with colchicine and hypotonic solution before fixation with Carnoy I solution. The results were 2nS = 24 (IIII+XiXt) in A. leucogramnui and P. velutina, and 2nS = 22 (10n+XiX2) in A. truncata, A. veniliae, P. kochi and Wagneriana sp. When the chromosomal morphologies were established, we observed telocentric chromosomes in all specimens save one female specimen of P. velutina. The univalent sex chromosomes were easily recognized on diplotenes. The unpaired metacentric element found in one female specimen of P. velutina with 2n = 25 probably arises by centric fusion/fission. Araneidae is a megadiverse family composed of -3000 species distributed mainly in the tropics; thus the analysis of more species may provide new insights about orb-weaver chromosome evolution.

O escorpião Tityus paraguayensis possui cromossomos holocêntricos, número diploide que varia de 2n=16 a 2n=18 e células em meiose I compostas apenas por bivalentes (8II; 9II) e/ou bivalentes mais associações multivalentes (5IICVII; 5IICVIII). Os estudos cromossômicos para esta espécie incluem apenas coloração convencional e localização de genes ribossomais e sequências teloméricas. Por meio da técnica de imunocitogenética, o objetivo deste trabalho foi investigar a presença de modificações epigenéticas nas histonas, tais como acetilação (H3K9ac e H4K5ac), fosforilação (H3S10f) e metilação (H3K4me2 e H3K9me2) ao longo da meiose bem como em estágios pós-meióticos. Testículos de T. paraguayensis foram fixados em paraformaldeído 2% e macerados em tampão 1xPBS. As lâminas foram incubadas com anticorpo primário para H3K9ac, H4K5ac, H3S10f, H3K4me2 e H3K9me2, e detectadas com anticorpo secundário anti-Rabbit conjugado com FITC. Os cromossomos foram contracorados com DAPI. A acetilação da lisina 9 da histona H3 ocorreu em núcleos interfásicos, enquanto a acetilação da lisina 5 da histona H4 foi observada em células pós-paquítênicas. Forte acetilação de histonas nas regiões organizadoras nucleolares permite que esses domínios cromossômicos evitem a condensação, na preparação para o início da transcrição que ocorre a partir da telófase. A dimetilação das lisinas 4 e 9 da H3  aconteceu em fases mais tardias da divisão celular, uma vez que a presença de tais anticorpos em fases iniciais não foi detectada. Metáfases mitóticas apresentaram cromossomos inteiramente hiperdimetilados com o uso do anticorpo H3K9me2. Não foi observado padrão de fosforilação da histona H3 em núcleos interfásicos. O nível de fosforilação é mínimo na interfase e aumenta progressivamente durante a divisão celular. Paquítenos e pós-paquítenos revelaram fosforilação dispersa ao longo dos cromossomos. Adicionalmente, as extremidades dos cromossomos apresentaram-se hiperfoforiladas. Tanto cromossomos holocêntricos quanto monocêntricos apresentam o mesmo padrão de fosforilação apesar de diferirem quanto à localização do cinetócoro. Este é o primeiro estudo relacionado a mudanças epigenéticas de histonas em escorpiões e permitiu descrever o padrão de tais modificações, as quais estão relacionadas à transcrição, condensação e segregação cromossômica de T. paraguayensis. Suporte Financeiro: FAPESP (Processo 2013/11840-0)