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Brachyhypopomus (Hypopomidae, Gymnotiformes) is a monophyletic genus consisting of 28 formally described species. Karyotypic data are available for 12 species. The same karyotype is described for two species ( B. brevirostris and B. hamiltoni ), as well as different karyotypes for the same species from distinct locations ( B. brevirostris ). In this context, B. brevirostris may constitute a cryptic species complex. Thus, in the present study, we analyzed the karyotype of B. brevirostris , from Santarém, Pará, and Tefé, Amazonas, using classical cytogenetics (conventional staining and C-banding) and molecular techniques (fluorescence in situ hybridization using 18S rDNA, 5S rDNA, U2 snRNA, and telomeric probes). The results show that samples from both locations present 2n = 38, with all chromosomes being acrocentric (FC = 38a). In both populations, 18S rDNA sequences are present on only one pair of homologous chromosomes and telomeric sequences occur only at the ends of the chromosomes. In the Tefé sample, the 5S rDNA occurs in two pairs, and the U2 snRNA in three pairs. These results are the first descriptions of these sequences for B. brevirostris samples from the Tefé locality, as well as the first karyotypic description for the Santarém locality. Future cytotaxonomic studies of this genus can benefit from these results.

Ipê is a plant of the Bignoniaceae family. Among the compounds extracted from this tree, lapachol is notable because its structural modification allows the production of β-lapachone, which has anticancer properties. The objective of this work was to test this hypothesis at a cellular level in vitro and assess its potential safety for use. The following tests were performed: MTT cell viability assay, apoptotic index determination, comet assay, and micronucleus test. The results showed that β-lapachone had a high cytotoxic capacity for all cell lines tested: ACP02 (gastric adenocarcinoma cells), MCF7 (breast carcinoma cells), HCT116 (colon cancer cells) and HEPG2 (hepatocellular carcinoma cells). Regarding genotoxicity, the exposure of cells to sublethal doses of β-lapachone induced DNA damage (assessed by the comet assay) and nuclear abnormalities, such as nucleoplasmic bridges and nuclear buds (assessed by the micronucleus test). All tested cell lines responded similarly to β-lapachone, except for ACP02 cells, which were relatively resistant to the cytotoxic effects of the compound in the MTT test. Our results collectively indicate that although β-lapachone showed antiproliferative activity against cancer cell lines, it also caused harmful effects in these cells, suggesting that the use of β-lapachone in treating cancer should be carried out with caution.

Rodents of the genus Cerradomys belong to tribe Oryzomyini, one of the most diverse and speciose groups in Sigmodontinae (Rodentia, Cricetidae). The speciation process in Cerradomys is associated with chromosomal rearrangements and biogeographic dynamics in South America during the Pleistocene era. As the morphological, molecular and karyotypic aspects of Myomorpha rodents do not evolve at the same rate, we strategically employed karyotypic characters for the construction of chromosomal phylogeny to investigate whether phylogenetic relationships using chromosomal data corroborate the radiation of Cerradomys taxa recovered by molecular phylogeny. Comparative chromosome painting using Hylaeamys megacephalus (HME) whole chromosome probes in C. langguthi (CLA), Cerradomys scotii (CSC), C. subflavus (CSU) and C. vivoi (CVI) shows that karyotypic variability is due to 16 fusion events, 2 fission events, 10 pericentric inversions and 1 centromeric repositioning, plus amplification of constitutive heterochromatin in the short arms of the X chromosomes of CSC and CLA. The chromosomal phylogeny obtained by Maximum Parsimony analysis retrieved Cerradomys as a monophyletic group with 97% support (bootstrap), with CSC as the sister to the other species, followed by a ramification into two clades (69% of branch support), the first comprising CLA and the other branch including CVI and CSU. We integrated the chromosome painting analysis of Eumuroida rodents investigated by HME and Mus musculus (MMU) probes and identified several syntenic blocks shared among representatives of Cricetidae and Muridae. The Cerradomys genus underwent an extensive karyotypic evolutionary process, with multiple rearrangements that shaped extant karyotypes. The chromosomal phylogeny corroborates the phylogenetic relationships proposed by molecular analysis and indicates that karyotypic diversity is associated with species radiation. Three syntenic blocks were identified as part of the ancestral Eumuroida karyotype (AEK): MMU 7/19 (AEK 1), MMU 14 (AEK 10) and MMU 12 (AEK 11). Besides, MMU 5/10 (HME 18/2/24) and MMU 8/13 (HME 22/5/11) should be considered as signatures for Cricetidae, while MMU 5/9/14, 5/7/19, 5 and 8/17 for Sigmodontinae., (© 2024. The Author(s).)

In the Amazon, some species of Loricariidae are at risk of extinction due to habitat loss and overexploitation by the ornamental fish market. Cytogenetic data related to the karyotype and meiotic cycle can contribute to understanding the reproductive biology and help management and conservation programs of these fish. Additionally, chromosomal mapping of repetitive DNA in Loricariidae may aid comparative genomic studies in this family. However, cytogenetics analysis is limited in Amazonian locariids. In this study, chromosomal mapping of multigenic families was performed in Scobinancistrus aureatus, Scobinancistrus pariolispos and Spatuloricaria sp. Meiotic analyzes were performed in Hypancistrus zebra and Hypancistrus sp. "pão". Results showed new karyotype for Spatuloricaria sp. (2n=66, NF=82, 50m-10sm-6m). Distinct patterns of chromosomal organization of histone H1, histone H3 and snDNA U2 genes were registered in the karyotypes of the studied species, proving to be an excellent cytotaxonomic tool. Hypotheses to explain the evolutionary dynamics of these sequences in studied Loricariidae were proposed. Regarding H. zebra and H. sp. "pão", we describe the events related to synapse and transcriptional activity during the meiotic cycle, which in both species showed 26 fully synapsed bivalents, with high gene expression only during zygotene and pachytene. Both Hypancistrus species could be used may be models for evaluating changes in spermatogenesis of Loricariidae., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Morphological, molecular and chromosomal studies in the genera Lonchothrix and Mesomys have contributed to a better understanding of taxonomic design, phylogenetic relationships and karyotypic patterns. Recent molecular investigations have shown a yet undescribed diversity, suggesting that these taxa are even more diverse than previously assumed. Furthermore, some authors have questioned the limits of geographic distribution in the Amazon region for the species M. hispidus and M. stimulax. In this sense, the current study sought to understand the karyotypic evolution and geographic limits of the genus Mesomys, based on classical (G- and C-banding) and molecular cytogenetic analysis (FISH using rDNA 18S and telomeric probes) and through the sequencing of mitochondrial genes Cytochrome b (Cytb) and Cytochrome Oxidase-Subunit I (CO using phylogeny, species delimitation and time of divergence, from samples of different locations in the Brazilian Amazon. The species M. stimulax and Mesomys sp. presented 2n = 60/FN = 110, while M. hispidus presented 2n = 60/FN = 112, hitherto unpublished. Molecular dating showed that Mesomys diversification occurred during the Plio-Pleistocene period, with M. occultus diverging at around 5.1 Ma, followed by Mesomys sp. (4.1 Ma) and, more recently, the separation between M. hispidus and M. stimulax (3.5 Ma). The ABGD and ASAP species delimiters support the formation of 7 and 8 potential species of the genus Mesomys, respectively. Furthermore, in both analyzes Mesomys sp. was recovered as a valid species. Our multidisciplinary approach involving karyotypic, molecular and biogeographic analysis is the first performed in Mesomys, with the description of a new karyotype for M. hispidus, a new independent lineage for the genus and new distribution data for M. hispidus and M. stimulax., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Dias de Oliveira et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Ancistrus is a highly diverse neotropical fish genus that exhibits extensive chromosomal variability, encompassing karyotypic morphology, diploid chromosome number (2n = 34-54), and the evolution of various types of sex chromosome systems. Robertsonian rearrangements related to unstable chromosomal sites are here described. Here, the karyotypes of two Ancistrus species were comparatively analyzed using classical cytogenetic techniques, in addition to isolation, cloning, sequencing, molecular characterization, and fluorescence in situ hybridization of repetitive sequences (i.e., 18S and 5S rDNA; U1, U2, and U5 snDNA; and telomere sequences). The species analyzed here have different karyotypes: Ancistrus sp. 1 (2n = 38, XX/XY) and Ancistrus cirrhosus (2n = 34, no heteromorphic sex chromosomes). Comparative mapping showed different organizations for the analyzed repetitive sequences: 18S and U1 sequences occurred in a single site in all populations of the analyzed species, while 5S and U2 sequences could occur in single or multiple sites. A sequencing analysis confirmed the identities of the U1, U2, and U5 snDNA sequences. Additionally, a syntenic condition for U2-U5 snDNA was found in Ancistrus . In a comparative analysis, the sequences of rDNA and U snDNA showed inter- and intraspecific chromosomal diversification. The occurrence of Robertsonian rearrangements and other dispersal mechanisms of repetitive sequences are discussed.

Malachite green (MG) is a synthetic dye that uses ranges from its application as a tissue dye to that as an antiparasitic in aquaculture. Several studies have reported the presence of this compound in food dyes and in the meat of fish raised in captivity for human consumption, suggesting risks both for the end user and for as those who handle these products because of MG toxic properties described in the literature. Here we evaluated the cytotoxic and genotoxic profiles of MG in four different cell lines (ACP02, L929, MNP01, and MRC-5). Two of these cell lines are stomach cells (normal and cancer lineages) and the potential ingestion of MG makes this a relevant cell type. Cells were treated with MG at concentrations ranging from 0.1 μM to 100 μM, and tested by MTT assay, a differential apoptosis/necrosis assay (EB/OA), the micronucleus test (MN), and the comet assay. MG exhibits dose-dependent cytotoxicity toward all of the tested cell types; higher concentrations of MG cause cell necrosis, while lower concentrations induce apoptosis. MG has a genotoxic profile increasing the rates of micronuclei, nucleoplasmic bridges, nuclear buds, and DNA fragmentation; L929 and MRC-5 showed more sensibility than ACP02 and MNP01., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© The Author(s) 2023. Published by Oxford University Press.)

The alkaloids isolated from Zanthoxylum rhoifolium have demonstrated great pharmacological potential; however, the toxic profiles of these extracts and fractions are still not well elucidated. This study evaluated the toxicity of the ethanol extract (EEZR) and neutral (FNZR) and alkaloid (FAZR) fractions. Chemical characterization was performed by chromatographic methods: thin-layer chromatography (TLC) and high-performance liquid chromatography coupled with diode array detection (HPLC-DAD). The cytotoxicity of the samples was evaluated in human hepatocellular carcinoma (HepG2) cells using the cell viability method (MTT) and mutagenicity by the Allium cepa assay (ACA). Alkaloids isolated from the species were selected for toxicity prediction using preADMET and PROTOX. The molecular docking of the topoisomerase II protein (TOPOII) was used to investigate the mechanism of cell damage. In the EEZR, FNZR, and FAZR, the presence of alkaloids was detected in TCL and HPLC-DAD analyses. These samples showed a 50% inhibitory concentration (IC 50 ) greater than 400 μg/mL in HepG2 cells. In ACA, time- and concentration-dependent changes were observed, with a significant reduction in the mitotic index and an increase in chromosomal aberrations for all samples. Nuclear sprouts and a micronucleus of the positive control (PC) were observed at 10 µg/mL and in the FAZR at 30 µg/mL; a chromosomal bridge in FNZR was observed at 105 µg/mL, CP at a concentration of 40 µg/mL, and nuclear bud and mitotic abnormalities in the EEZR were observed at 170 µg/mL. The alkaloids with a benzophenanthridine were selected for the in silico study, as structural alterations demonstrated certain toxic effects. Molecular docking with topo II demonstrated that all alkaloids bind to the protein. In summary, the fractionation of Z. rhoifolium did not interfere with toxicity; it seems that alkaloids with a benzophenanthridine nucleus may be involved in this toxicity.

Docosahexaenoic acid (DHA), also known as omega-3 ( n -3) polyunsaturated fatty acid (PUFA), is a natural compound that has demonstrated pharmacological activity against several malignant neoplasms. Available cancer treatments cause side effects, affect healthy cells, reduce the quality of life of patients and may cause resistance to antineoplastics. For these reasons, the search for new therapies is continuous. This narrative review aimed to compile information on in vitro experiments that study the cytotoxic effect of DHA or molecules derived from DHA in tumor and nontumor cells. This was performed to highlight the potential of DHA as a strategy for cancer therapy and to gather information, which will help researchers plan experimental designs and develop research to discover effective therapies against cancer. In addition, studies were presented that demonstrate the dose of DHA that can treat patients with cancer. Thus, a search was conducted for articles on the SCOPUS and Web of Science platforms, published until 2022, that analyzed the action of DHA against breast, lung, colorectal, prostate, stomach and liver cancers. Cytotoxic effects were observed in tumor and nontumor cell lines, and these results varied with the type of cell line studied, drug concentration, incubation time and treatment combination, i.e., with DHA alone, combined with other drugs and with molecules derived from DHA. In patients with cancer, in all analyzed studies, DHA intake was associated with eicosapentaenoic acid (EPA) and/or proteins to aid chemotherapy, and with this procedure, tumor reduction, chemotherapy tolerance and muscle mass gain were obtained. This work contributes to the community by demonstrating the possible applicability of DHA in the pharmaceutical area of oncological therapies.

Loricariidae (Siluriformes) comprises ∼1026 species of neotropical fish, being considered the most diverse among the Siluriformes. Studies on repetitive DNA sequences have provided important data on the evolution of the genomes of members of this family, especially of the Hypostominae subfamily. In this study, the chromosomal mapping of the histone multigene family and U2 snRNA was performed in two species belonging to the Hypancistrus genus, Hypancistrus sp. "pão" (2n = 52, 22m + 18sm +12st) and Hypancistrus zebra (2n = 52, 16m + 20sm +16st). The presence of dispersed signals of histones H2A, H2B, H3, and H4 in the karyotype of both species, with each sequence displaying a varied level of accumulation and dispersion of these sequences between them was observed; in addition, U2 snDNA probe only showed positive results in H. zebra , which present this multigene in the terminal region of three chromosomal pairs. The obtained results resemble data already analyzed in the literature, in which the action of transposable elements interfere in the organization of these multigene families, in addition to other evolutionary processes that shape the evolution of the genome, such as circular or ectopic recombination. This study also shows that the dispersion of the multigene histone family is quite complex, and from this, these data serve as a point of discussion for the evolutionary processes that occur in the Hypancistrus karyotype.

Background: Chromosomal painting in manatees has clarified questions about the rapid evolution of sirenians within the Paenungulata clade. Further cytogenetic studies in Afrotherian species may provide information about their evolutionary dynamics, revealing important insights into the ancestral karyotype in the clade representatives. The karyotype of Trichechus inunguis (TIN, Amazonian manatee) was investigated by chromosome painting, using probes from Trichechus manatus latirostris (TML, Florida manatee) to analyze the homeologies between these sirenians., Results: A high similarity was found between these species, with 31 homologous segments in TIN, nineteen of which are whole autosomes, besides the X and Y sex chromosomes. Four chromosomes from TML (4, 6, 8, and 9) resulted in two hybridization signals, totaling eight acrocentrics in the TIN karyotype. This study confirmed in TIN the chromosomal associations of Homo sapiens (HSA) shared in Afrotheria, such as the 5/21 synteny, and in the Paenungulata clade with the syntenies HSA 2/3, 8/22, and 18/19, in addition to the absence of HSA 4/8 common in eutherian ancestral karyotype (EAK)., Conclusions: TIN shares more conserved chromosomal signals with the Paenungulata Ancestral Karyotype (APK, 2n = 58) than Procavia capensis (Hyracoidea), Loxodonta africana (Proboscidea) and TML (Sirenia), where TML presents less conserved signals with APK, demonstrating that its karyotype is the most derived among the representatives of Paenungulata. The chromosomal changes that evolved from APK to the T. manatus and T. inunguis karyotypes (7 and 4 changes, respectively) are more substantial within the Trichechus genus compared to other paenungulates. Among these species, T. inunguis presents conserved traits of APK in the American manatee genus. Consequently, the karyotype of T. manatus is more derived than that of T. inunguis., (© 2023. The Author(s).)

Charadriidae comprise 142 valid species and the most recent checklist for the occurrence of this family in Brazil describes 11 species. There are few chromosomal studies in Charadriidae, most of them using a conventional approach. In Charadrius, only five species had their karyotypes described by classical cytogenetics, of which four have 2n = 76 (C. hiaticula, C. dubius, C. vociferou and C. collaris) and one 2n = 78 (C. alexandrinus alexandrinus). Among these species, only Charadrius collaris had the karyotype studied by chromosome painting, which allowed the identification of chromosomal homeologies with the karyotypes of Gallus gallus (GGA) and Burhinus oedicnemus (BOE). According to the literature, studies performed with BAC-FISH using probes from Gallus gallus and Taeniopygia guttata (TGU) libraries have shown interactions between macro and microchromosomes and micro inversions in chromosomes previously considered conserved. Other studies have shown the fusion of several microchromosomes, forming new macrochromosomes, leading to a decrease in the 2n of some species. The present study aims to deepen the chromosomal information in Charadrius collaris through the application of BAC-FISH with probes from the GGA and TGU libraries, in order to investigate possible rearrangements within the apparently conserved karyotype of this species, and thus better clarify the evolutionary history of the species. Charadrius collaris presented 2n = 76 and fundamental number (FN) equal to 94. Comparative mapping of BAC probes from GGA and TGU in Charadrius collaris revealed hybridization signals from 26 macrochromosome probes. Probes from microchromosomes 9 to 28 of GGA were also used and revealed 31 hybridization signals. The karyotype is well conserved, but it contains a paracentric and a pericentric inversion on the CCO1 chromosome, a paracentric and a pericentric inversion on the CCO4 and the separation of GGA4 into CCO4 and CCO8, demonstrating that the BAC-FISH approach allows for greater data resolution. More studies are needed to improve the understanding of chromosomal evolution within the order Charadriiformes and thus clarify whether these characteristics demonstrated here are specific traits for Charadrius collaris or if other species share these characteristics., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Ferreira et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The Croton cajucara (Euphorbiales, Euphorbiaceae) plant occurs widely in the Amazon region, where its leaves and stem bark are consumed by the population to treat several diseases. The secondary metabolite trans-dehydrocrotonin (DCTN) is mainly accountable for the biological activities of this plant. However, prolonged consumption is associated with hepatotoxic effects due to the furan ring present in the molecule. This group is responsible for toxicity reactions in other drugs. In this work, we inserted a COOH group into the molecule to prevent the formation of toxic intermediates. We assessed the cytotoxicity and genotoxicity of both molecules on HepG2 cells. Results showed that the new derivative (CCTN) is less cytotoxic and did not reduce cell viability at any concentration tested. Genotoxicity was also reduced as assessed by the comet and micronucleus assay. Therefore, the new derivative appears to be promising and additional tests should be performed to evaluate its therapeutic activities., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The subfamily Phyllostominae (Chiroptera, Phyllostomidae) comprises 10 genera of Microchiroptera bats from the Neotropics. The taxonomy of this group is controversial due to incongruities in the phylogenetic relationships evident from different datasets. The genus Lophostoma currently includes eight species whose phylogenetic relationships have not been resolved. Integrative analyzes including morphological, molecular and chromosomal data are powerful tools to investigate the phylogenetics of organisms, particularly if obtained by chromosomal painting. In the present work we performed comparative genomic mapping of three species of Lophostoma (L. brasiliense 2n = 30, L. carrikeri 2n = 26 and L. schulzi 2n = 26), by chromosome painting using whole chromosome probes from Phyllostomus hastatus and Carollia brevicauda; this included mapping interstitial telomeric sites. The karyotype of L. schulzi (LSC) is a new cytotype. The species L. brasiliense and L. carrikeri showed interstitial telomeric sequences that probably resulted from expansions of repetitive sequences near pericentromeric regions. The addition of chromosomal painting data from other species of Phyllostominae allowed phylogeny construction by maximum parsimony, and the determination that the genera of this subfamily are monophyletic, and that the genus Lophostoma is paraphyletic. Additionally, a review of the taxonomic status of LSC is suggested to determine if this species should be reclassified as part of the genus Tonatia., (© 2022. The Author(s).)

Pseudacanthicus is a genus of Neotropical fish with eight valid species, in addition to numerous lineages not formally identified. It occurs along the Amazon and Tocantins River basins, in Suriname and in the Guiana shield. There are no karyotypic data in the literature for species of this genus. Here, the karyotypes of three Pseudacanthicus species ( P. spinosus , P. leopardus and Pseudacanthicus sp.) were comparatively analyzed by classical cytogenetics and fluorescence in situ hybridization using 18S and 5S rDNA probes, U2 snDNA and telomeric sequences. The analyzed species presented 52 chromosomes and KF = 18 m + 34 sm. Constitutive heterochromatin occurred in blocks on a few chromosomes. The 18S rDNA occurred in a single pair; interestingly, P. leopardus presented only one locus of this sequence in its diploid genome. The 5S rDNA sequence occurred in only one pair in P. leopardus , and in multiple sites in Pseudacanthicus sp. and P. spinosus . The snDNA U2 occurred in only one pair in all analyzed species. Telomeric sequences did not show interstitial sites. Although Pseudacanthicus species share the same 2n and KF, repetitive sequence analysis revealed karyotypic diversity among these species. The occurrence of DNA double-strand breaks related to fragile sites, unequal crossing over and transpositions is proposed as the mechanism of karyotypic diversification, suggesting that the conservation of the karyotypic macrostructure is only apparent in this group of fish.

Several species of Tityus (Scorpiones, Buthidae) present multi-chromosomal meiotic associations and failures in the synaptic process, originated from reciprocal translocations. Holocentric chromosomes and achiasmatic meiosis in males are present in all members of this genus. In the present study, we investigated synapse dynamics, transcriptional silencing by γH2AX, and meiotic microtubule association in bivalents and a quadrivalent of the scorpion Tityus maranhensis . Additionally, we performed RT-PCR to verify the expression of mismatch repair enzymes involved in crossing-over formation in Tityus silvestris gonads. The quadrivalent association in T. maranhensis showed delay in the synaptic process and long asynaptic regions during pachytene. In this species, γH2AX was recorded only at the chromosome ends during early stages of prophase I; in metaphase I, bivalents and quadrivalents of T. maranhensis exhibited binding to microtubules along their entire length, while in metaphase II/anaphase II transition, spindle fibers interacted only with telomeric regions. Regarding T. silvestris , genes involved in the recombination process were transcribed in ovaries, testes and embryos, without significant difference between these tissues. The expression of these genes during T. silvestris achiasmatic meiosis is discussed in the present study. The absence of meiotic inactivation by γH2AX and holo/telokinetic behavior of the chromosomes are important factors for the maintenance of the quadrivalent in T. maranhensis and the normal continuation of the meiotic cycle in this species.

Charadriiformes represent one of the largest orders of birds; members of this order are diverse in morphology, behavior and reproduction, making them an excellent model for studying evolution. It is accepted that the avian putative ancestral karyotype, with 2n = 80, remains conserved for about 100 million years. So far, only a few species of Charadriiformes have been studied using molecular cytogenetics. Here, we performed chromosome painting on metphase chromosomes of two species of Charadriidae, Charadrius collaris and Vanellus chilensis, with whole chromosome paint probes from Burhinus oedicnemus. Charadrius collaris has a diploid number of 76, with both sex chromosomes being submetacentric. In V. chilensi a diploid number of 78 was identified, and the Z chromosome is submetacentric. Chromosome painting suggests that chromosome conservation is a characteristic common to the family Charadriidae. The results allowed a comparative analysis between the three suborders of Charadriiformes and the order Gruiformes using chromosome rearrangements to understand phylogenetic relationships between species and karyotypic evolution. However, the comparative analysis between the Charadriiformes suborders so far has not revealed any shared rearrangements, indicating that each suborder follows an independent evolutionary path, as previously proposed. Likewise, although the orders Charadriiformes and Gruiformes are placed on sister branches, they do not share any signature chromosomal rearrangements., Competing Interests: The authors have declared that no competing interests exist.

Great efforts have been made to preserve manatees. Recently, a hybrid zone was described between Trichechus inunguis (TIN) and the Trichechus manatus manatus (TMM) in the Amazon estuary. Cytogenetic data on these sirenians are limited, despite being fundamental to understanding the hybridization/introgression dynamics and genomic organization in Trichechus . We analyzed the karyotype of TMM, TIN, and two hybrid specimens ("Poque" and "Vitor") by classical and molecular cytogenetics. G-band analysis revealed that TMM (2n = 48) and TIN (2n = 56) diverge by at least six Robertsonian translocations and a pericentric inversion. Hybrids had 2n = 50, however, with Autosomal Fundamental Number (FNA) = 88 in "Poque" and FNA = 74 in "Vitor", and chromosomal distinct pairs in heterozygous; additionally, "Vitor" exhibited heteromorphisms and chromosomes whose pairs could not be determined. The U2 snDNA and Histone H3 multi genes are distributed in small clusters along TIN and TMM chromosomes and have transposable Keno and Helitron elements (TEs) in their sequences. The different karyotypes observed among manatee hybrids may indicate that they represent different generations formed by crossing between fertile hybrids and TIN. On the other hand, it is also possible that all hybrids recorded represent F1 and the observed karyotype differences must result from mechanisms of elimination.

X-autosome translocation (XY 1 Y 2 ) has been reported in distinct groups of vertebrates suggesting that the rise of a multiple sex system within a species may act as a reproductive barrier and lead to speciation. The viability of this system has been linked with repetitive sequences located between sex and autosomal portions of the translocation. Herein, we investigate Oecomys auyantepui, using chromosome banding and Fluorescence In Situ Hybridization with telomeric and Hylaeamys megacephalus whole-chromosome probes, and phylogenetic reconstruction using mtDNA and nuDNA sequences. We describe an amended karyotype for O. auyantepui (2n = 64♀65♂/FNa = 84) and report for the first time a multiple sex system (XX/XY 1 Y 2 ) in Oryzomyini rodents. Molecular data recovered O. auyantepui as a monophyletic taxon with high support and cytogenetic data indicate that O. auyantepui may exist in two lineages recognized by distinct sex systems. The Neo-X exhibits repetitive sequences located between sex and autosomal portions, which would act as a boundary between these two segments. The G-banding comparisons of the Neo-X chromosomes of other Sigmodontinae taxa revealed a similar banding pattern, suggesting that the autosomal segment in the Neo-X can be shared among the Sigmodontinae lineages with a XY 1 Y 2 sex system., (© 2022. The Author(s).)

The genus Gymnotus is a large monophyletic group of freshwater weakly-electric fishes, with wide distribution in Central and South America. It has 46 valid species divided into six subgenera ( Gymnotus , Tijax , Tigre , Lamontianus , Tigrinus and Pantherus ) with large chromosome plasticity and diploid numbers (2n) ranging from 34 to 54. Within this rich diversity, there is controversy about whether Gymnotus ( Gymnotus ) carapo species is a single widespread species or a complex of cryptic species. Cytogenetic studies show different diploid numbers for G. carapo species, ranging from 40 to 54 chromosomes with varied karyotypes found even between populations sharing the same 2n. Whole chromosome painting has been used in studies on fish species and recently has been used for tracking the chromosomal evolution of Gymnotus and assisting in its cytotaxonomy. Comparative genomic mapping using chromosome painting has shown more complex rearrangements in Gymnotus carapo than shown in previous studies by classical cytogenetics. These studies demonstrate that multiple chromosome pairs are involved in its chromosomal reorganization , suggesting the presence of a complex of cryptic species due to a post zygotic barrier. In the present study, metaphase chromosomes of G. carapo occidentalis "catalão" (GCC, 2n = 40, 30m/sm+10st/a) from the Catalão Lake, Amazonas, Brazil, were hybridized with whole chromosome probes derived from the chromosomes of G. carapo (GCA, 2n = 42, 30m/sm+12st/a). The results reveal chromosome rearrangements and a high number of repetitive DNA sites. Of the 12 pairs of G. carapo chromosomes that could be individually identified (GCA 1-3, 6, 7, 9, 14, 16 and 18-21), 8 pairs (GCA 1, 2, 6, 7, 9, 14, 20, 21) had homeology conserved in GCC. Of the GCA pairs that are grouped (GCA [4, 8], [5, 17], [10, 11] and [12, 13, 15]), most kept the number of signals in GCC (GCA [5, 17], [10, 11] and [12, 13, 15]). The remaining chromosomes are rearranged in the GCC karyotype. Analysis of both populations of the G. carapo cytotypes shows extensive karyotype reorganization. Along with previous studies, this suggests that the different cytotypes analyzed here may represent different species and supports the hypothesis that G. carapo is not a single widespread species, but a group of cryptic species., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Machado, da Silva, Feldberg, O’Brien, Ferguson-Smith, Pieczarka and Nagamachi.)

The Ancistrus genus has extensive chromosomal diversity among species, including heteromorphic sex chromosomes occurrence. However, studies have been shown that chromosomal diversity may still be underestimated. Repetitive sequences represent a large part of eukaryotic genomes, associated with mechanisms of karyotypic diversification, including sex chromosomes evolution. This study analyzed the karyotype diversification of two Ancistrus species ( Ancistrus sp. 1 and Ancistrus sp. 2) from the Amazon region by classical and molecular chromosomal markers. Conventional chromosome bands and fluorescence in situ hybridization using probes 18S and 5S rDNA, besides (CA)n, (CG)n, (GA)n, (CAC)n, (CAG)n, (CAT)n, (GAA)n, (GAC)n, (TAA)n, and (TTAGGG)n in tandem repeats were determined on the karyotypes. Ancistrus sp. 1 and Ancistrus sp. 2 presented karyotypes with 2n = 38 (20 m + 14sm+4st, XX/XY) and 2n = 34 (20 m + 14sm, without heteromorphic sex chromosomes), respectively. Robertsonian rearrangements can explain the diploid number difference. C-bands occurred in pericentromeric regions in some chromosomes, and a single 18S rDNA locus occurred in both species. The 5S rDNA showed variation in the number of loci between species karyotypes, suggesting the occurrence of unstable sites and rearrangements associated with these sequences in Ancistrus. The microsatellite mapping evidenced distinct patterns of organization between the two analyzed species, occurring mainly in the sex chromosomes in Ancistrus sp. 1, and in the centromeric and pericentromeric regions of chromosomes m/sm in Ancistrus sp. 2. These data shows the extensive chromosomal diversity of repetitive sequences in Ancistrus , which were involved in Robertsonian rearrangements and sex chromosomes differentiation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Santos da Silva, Glugoski, Vicari, de Souza, Noronha, Pieczarka and Nagamachi.)

Archolaemus , one of the five genera of Neotropical freshwater fish of the family Sternopygidae (Gymnotiformes), was long considered a monotypic genus represented by Archolaemus blax . Currently, it consists of six species, most of them occurring in the Amazon region. There are no cytogenetic data for species of this genus. In the present study, we used classical cytogenetics (conventional staining and C-banding) and molecular cytogenetics (probes of telomeric sequences and multigenic families 18S rDNA, 5S rDNA, and U2 snDNA) to study the karyotype of Archolaemus janeae from Xingu and Tapajós rivers in the state of Pará (Brazil). The results showed that the two populations have identical karyotypes with 46 chromosomes: four submetacentric and 42 acrocentric (2 n  = 46; 4m/sm + 42a). Constitutive heterochromatin occurs in the centromeric region of all chromosomes, in addition to small bands in the interstitial and distal regions of some pairs. The 18S rDNA occurs in the distal region of the short arm of pair 2; the 5S rDNA occurs in five chromosome pairs; and the U2 snDNA sequence occurs in chromosome pairs 3, 6, and 13. No interstitial telomeric sequence was observed. These results show karyotypic similarity between the studied populations suggesting the existence of a single species and are of great importance as a reference for future cytotaxonomic studies of the genus., Competing Interests: The authors declare that there is no conflict of interest., (© 2021 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

Peckoltia is widely distributed genus in the Amazon and Orinoco basins and the Guiana Shield, containing 18 valid species, and distinct morphotypes still needing description in the scientific literature due to its great taxonomic complexity. This study performed a comparative chromosomal analysis of two undescribed Peckoltia species ( Peckoltia sp. 3 Jarumã and Peckoltia sp. 4 Caripetuba) from the Brazilian Amazon using conventional chromosome bands methods and in situ localization of the repetitive DNA ( 5S and 18S rRNA and U1 snRNA genes and telomeric sequences). Both species presented 2n = 52 but differed in their karyotype formula, probably due to inversions or translocations. The nucleolus organizer regions (NORs) showed distal location on a probably homeologous submetacentric pair in both species, besides an extra signal in a subtelocentric chromosome in Peckoltia sp. 4 Caripetuba. Heterochromatin occurred in large blocks, with different distributions in the species. The mapping of the 18S and 5S rDNA, and U1 snDNA showed differences in locations and number of sites. No interstitial telomeric sites were detected using the (TTAGGG)n probes. Despite 2n conservationism in Peckoltia species, the results showed variation in karyotype formulas, chromosomal bands, and locations of repetitive sites, demonstrating great chromosomal diversity. A proposal for Peckoltia karyotype evolution was inferred in this study based on the diversity of location and number of chromosomal markers analyzed. A comparative analysis with other Peckoltia karyotypes described in the literature, their biogeography patterns, and molecular phylogeny led to the hypothesis that the derived karyotype was raised in the left bank of the Amazon River., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Santos da Silva, de Souza, Pety, Noronha, Vicari, Pieczarka and Nagamachi.)

Rhipidomys (Sigmodontinae, Thomasomyini) has 25 recognized species, with a wide distribution ranging from eastern Panama to northern Argentina. Cytogenetic data has been described for 13 species with 12 of them having 2n = 44 with a high level of autosomal fundamental number (FN) variation, ranging from 46 to 80, assigned to pericentric inversions. The species are grouped in groups with low FN (46-52) and high FN (72-80). In this work the karyotypes of Rhipidomys emiliae (2n = 44, FN = 50) and Rhipidomys mastacalis (2n = 44, FN = 74), were studied by classical cytogenetics and by fluorescence in situ hybridization using telomeric and whole chromosome probes (chromosome painting) of Hylaeamys megacephalus (HME). Chromosome painting revealed homology between 36 segments of REM and 37 of RMA. We tested the hypothesis that pericentric inversions are the predominant chromosomal rearrangements responsible for karyotypic divergence between these species, as proposed in literature. Our results show that the genomic diversification between the karyotypes of the two species resulted from translocations, centromeric repositioning and pericentric inversions. The chromosomal evolution in Rhipidomys was associated with karyotypical orthoselection. The HME probes revealed that seven syntenic probably ancestral blocks for Sigmodontinae are present in Rhipidomys. An additional syntenic block described here is suggested as part of the subfamily ancestral karyotype. We also define five synapomorphies that can be used as chromosomal signatures for Rhipidomys., Competing Interests: The authors have declared that no competing interests exist.

Animal models represent a crucial tool for biological research, so the establishment of new cultures is fundamental for the discovery of new therapies and the understanding of mechanisms of cell development in the most diverse animals. Here, we report the successful establishment of two new primary cell cultures derived from a South American bat (Artibeus planirostris). The establishment of a new bat culture can help in the investigation of new zoonoses since bats have been proposed as carriers of these diseases. We evaluated the chromosomal stability of cells from different passages. Primary cultures were collected from ear tissues and bone marrow of A. planirostris. Cultures were expanded, and osteogenic and adipogenic inductions were conducted for 21 days. For osteogenic differentiation, the medium was supplemented with 0.1 μM dexamethasone, 3 mM β-glycerophosphate, and 10 μM L-ascorbic acid 2-phosphate. For adipogenic differentiation, the medium was supplemented with 5 μM rosiglitazone, 0.4 μM insulin, 0.1 mM indomethacin, and 0.1 μM dexamethasone. After the induction period, the cells were stained with Alizarin Red to assess osteogenic differentiation and Oil Red O to assess adipogenic differentiation. We observed the appearance of lipid droplets in adipocytes and the extracellular deposition of calcium matrix by osteocytes, indicating that bone marrow-derived cells and skin-derived cells of A. planirostris could successfully differentiate into these lineages. Also, the number of chromosomes remained stable for both primary cultures during passages 2, 4, 6, and 8., (Copyright © 2021. Published by Elsevier Ltd.)

Doxorubicin (DOX) is an anthracycline antibiotic used in the fight against many types of cancer. Although it is quite effective for this purpose, its clinical use is limited by its severe side effects, highlighting the relevance of efforts to identify substances that act to minimize these effects. In this work, we sought to verify the ability of andiroba oil (AO) and a nanoemulsion of andiroba oil (AN) to lessen the side effects of DOX. The animals were separated into 7 groups with 6 animals each: mice treated with AO (2000 mg/kg), AN (2000 mg/kg), the antineoplastic agent DOX (40 mg/kg), AO+DOX, AN+DOX and solvent controls was used of negative control (corn oil and nanoemulsion surfactant). AO and AN were administered for 14 consecutive days orally by gavage and on the 13th day, applied DOX by intraperitoneal route (i.p.), in order to evaluate the protective potential of andiroba. The animals were euthanized on the 15th day. Hematological, biochemical, histological, and immunohistochemical parameters were analyzed. Andiroba reduced several aspects of the severity of lesions caused by DOX, decreasing hematotoxicity and the severity of histological changes in the liver and kidneys, and reducing the frequency of apoptotic cell death. In many cases, AN showed greater efficacy than AO alone, reflecting the feasibility of using this nanotechnology to improve the pharmacokinetics of lipid compounds in the body. The study sheds new light on the therapeutic benefits of andiroba and suggests new ways for investigating how the quantity and quality of lipid compounds affect exposed organisms., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Mesomys Wagner, 1845 (Rodentia, Echimyidae, Eumysopinae) currently has four recognized species, three of which occur in Brazil: Mesomys hispidus (probably a species complex), M .  occultus, and M .  stimulax . Mesomys leniceps is found in montane forests of northern Peru. Mesomys stimulax , the focus of the present study, has a distribution that is restricted to the central and eastern Amazonia south of the Amazon River, extending from the left bank of the Tapajós River to the right bank of the Tocantins River, and south to the southeast portion of Pará State. The genus presents karyotypes with diploid number 2n = 60 and Fundamental Number (FN) = 116 for M .  hispidus and M .  stimulax , and 2n = 42, FN = 54 for M .  occultus . We studied the karyotype of a female specimen of M .  stimulax collected from the Tapirapé-Aquiri National Forest, Marabá, Pará, Brazil, in the Xingu/Tocantins interfluvium. The obtained karyotype (2n = 60 and FN = 110) differs from that described in the literature for both M .  stimulax and M .  hispidus by exhibiting more biarmed chromosomes, probably due to pericentric inversions and/or centromeric repositioning, and exhibiting differences in the amount and distribution of constitutive heterochromatin (CH). These results suggest that, similar to what has already been proposed for M .  hispidus , M .  stimulax may represent a species complex and/or cryptic species. The mechanisms of chromosomal diversification in Mesomys and the biogeographic implications are discussed reinforcing the need for broad systematic review for Mesomys ., Competing Interests: The authors declare no conflict of interest., (© 2021 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

The use of clean technologies in the development of bioactive plant extracts has been encouraged, but it is necessary to verify the cytotoxicity and cytoprotection for food and pharmaceutical applications. Therefore, the objective of this work was to obtain the experimental data of the supercritical sequential extraction of murici pulp, to determine the main bioactive compounds obtained and to evaluate the possible cytotoxicity and cytoprotection of the extracts in models of HepG2 cells treated with H 2 O 2 . The murici pulp was subjected to sequential extraction with supercritical CO 2 and CO 2 +ethanol, at 343.15 K, and 22, 32, and 49 MPa. Higher extraction yields were obtained at 49 MPa. The oil presented lutein (224.77 µg/g), oleic, palmitic, and linoleic, as the main fatty acids, and POLi (17.63%), POO (15.84%), PPO (13.63%), and LiOO (10.26%), as the main triglycerides. The ethanolic extract presented lutein (242.16 µg/g), phenolic compounds (20.63 mg GAE/g), and flavonoids (0.65 mg QE/g). The ethanolic extract showed greater antioxidant activity (122.61 and 17.14 µmol TE/g) than oil (43.48 and 6.04 µmol TE/g). Both extracts did not show cytotoxicity and only murici oil showed a cytoprotective effect. Despite this, the results qualify both extracts for food/pharmaceutical applications.

Background: Thamnophilidae birds are the result of a monophyletic radiation of insectivorous Passeriformes. They are a diverse group of 225 species and 45 genera and occur in lowlands and lower montane forests of Neotropics. Despite the large degree of diversity seen in this family, just four species of Thamnophilidae have been karyotyped with a diploid number ranging from 76 to 82 chromosomes. The karyotypic relationships within and between Thamnophilidae and another Passeriformes therefore remain poorly understood. Recent studies have identified the occurrence of intrachromosomal rearrangements in Passeriformes using in silico data and molecular cytogenetic tools. These results demonstrate that intrachromosomal rearrangements are more common in birds than previously thought and are likely to contribute to speciation events. With this in mind, we investigate the apparently conserved karyotype of Willisornis vidua, the Xingu Scale-backed Antbird, using a combination of molecular cytogenetic techniques including chromosome painting with probes derived from Gallus gallus (chicken) and Burhinus oedicnemus (stone curlew), combined with Bacterial Artificial Chromosome (BAC) probes derived from the same species. The goal was to investigate the occurrence of rearrangements in an apparently conserved karyotype in order to understand the evolutionary history and taxonomy of this species. In total, 78 BAC probes from the Gallus gallus and Taeniopygia guttata (the Zebra Finch) BAC libraries were tested, of which 40 were derived from Gallus gallus macrochromosomes 1-8, and 38 from microchromosomes 9-28., Results: The karyotype is similar to typical Passeriformes karyotypes, with a diploid number of 2n = 80. Our chromosome painting results show that most of the Gallus gallus chromosomes are conserved, except GGA-1, 2 and 4, with some rearrangements identified among macro- and microchromosomes. BAC mapping revealed many intrachromosomal rearrangements, mainly inversions, when comparing Willisornis vidua karyotype with Gallus gallus, and corroborates the fissions revealed by chromosome painting., Conclusions: Willisornis vidua presents multiple chromosomal rearrangements despite having a supposed conservative karyotype, demonstrating that our approach using a combination of FISH tools provides a higher resolution than previously obtained by chromosome painting alone. We also show that populations of Willisornis vidua appear conserved from a cytogenetic perspective, despite significant phylogeographic structure.

Background: The Scolopacidae family (Suborder Scolopaci, Charadriiformes) is composed of sandpipers and snipes; these birds are long-distance migrants that show great diversity in their behavior and habitat use. Cytogenetic studies in the Scolopacidae family show the highest diploid numbers for order Charadriiformes. This work analyzes for the first time the karyotype of Actitis macularius by classic cytogenetics and chromosome painting., Results: The species has a diploid number of 92, composed mostly of telocentric pairs. This high 2n is greater than the proposed 80 for the avian ancestral putative karyotype (a common feature among Scolopaci), suggesting that fission rearrangements have formed smaller macrochromosomes and microchromosomes. Fluorescence in situ hybridization using Burhinus oedicnemus whole chromosome probes confirmed the fissions in pairs 1, 2, 3, 4 and 6 of macrochromosomes., Conclusion: Comparative analysis with other species of Charadriiformes studied by chromosome painting together with the molecular phylogenies for the order allowed us to raise hypotheses about the chromosomal evolution in suborder Scolopaci. From this, we can establish a clear idea of how chromosomal evolution occurred in this suborder.

The genus Oecomys (Rodentia, Sigmodontinae) is distributed from southern Central America to southeastern Brazil in South America. It currently comprises 18 species, but multidisciplinary approaches such as karyotypic, morphological and molecular studies have shown that there is a greater diversity within some lineages than others. In particular, it has been proposed that O. paricola constitutes a species complex with three evolutionary units, which have been called the northern, eastern and western clades. Aiming to clarify the taxonomic status of O. paricola and determine the relevant chromosomal rearrangements, we investigated the karyotypes of samples from eastern Amazonia by chromosomal banding and FISH with Hylaeamys megacephalus (HME) whole-chromosome probes. We detected three cytotypes for O. paricola: A (OPA-A; 2n = 72, FN = 75), B (OPA-B; 2n = 70, FN = 75) and C (OPA-C; 2n = 70, FN = 72). Comparative chromosome painting showed that fusions/fissions, translocations and pericentric inversions or centromeric repositioning were responsible for the karyotypic divergence. We also detected exclusive chromosomal signatures that can be used as phylogenetic markers. Our analysis of karyotypic and distribution information indicates that OPA-A, OPA-B and OPA-C are three distinct species that belong to the eastern clade, with sympatry occurring between two of them, and that the "paricola group" is more diverse than was previously thought., Competing Interests: The authors have declared that no competing interests exist.

Heterozygous chromosomal rearrangements can result in failures during the meiotic cycle and the apoptosis of germline, making carrier individuals infertile. The Amazon frog Leptodactylus pentadactylus has a meiotic multivalent, composed of 12 sex chromosomes. The mechanisms by which this multi-chromosome system maintains fertility in males of this species remain undetermined. In this study we investigated the meiotic behavior of this multivalent to understand how synapse, recombination and epigenetic modifications contribute to maintaining fertility and chromosomal sexual determination in this species. Our sample had 2n = 22, with a ring formed by ten chromosomes in meiosis, indicating a new system of sex determination for this species (X1Y1X2Y2X3Y3X4Y4X5Y5). Synapsis occurs in the homologous terminal portion of the chromosomes, while part of the heterologous interstitial regions performed synaptic adjustment. The multivalent center remains asynaptic until the end of pachytene, with interlocks, gaps and rich-chromatin in histone H2A phosphorylation at serine 139 (γH2AX), suggesting transcriptional silence. In late pachytene, paired regions show repair of double strand-breaks (DSBs) with RAD51 homolog 1 (Rad51). These findings suggest that Rad51 persistence creates positive feedback at the pachytene checkpoint, allowing meiosis I to progress normally. Additionally, histone H3 trimethylation at lysine 27 in the pericentromeric heterochromatin of this anuran can suppress recombination in this region, preventing failed chromosomal segregation. Taken together, these results indicate that these meiotic adaptations are required for maintenance of fertility in L. pentadactylus.

Malachite green (MG) is a triphenylmethane dye that is widely used in aquaculture as a fungicide, bactericide, ectoparasiticide, and antiprotozoal. There is great debate regarding the potential for this compound to trigger adverse effects. Here, we review the previous findings and then evaluate the lethal and sublethal effects of MG in the species Hemichromis bimaculatus (jewelfish). The lethal concentration for 50% of the fish in 96 h was 1 mg/L. We observed a dose-dependent increase in the percentage of fish mortality as well as physical and behavioral changes. We further found that the highest tested sublethal dose significantly increased the DNA damage index identified using the comet assay (74.97 ± 13.8 at a significant level of P < 0.05 for the 0.75 mg/L concentration), but did not significantly alter the results of the micronucleus test. Although our results suggest that MG confers risks on exposed fish, the findings were significant only at the highest exposure concentration (0.75 mg/L). At lower concentrations (0.25 mg/L and 0.5 mg/L), no adverse effect was observed. The maximum MG concentration recommended for use in ornamental fish farming is 0.2 mg/L. Therefore, our results suggest that, specifically for the parameters analyzed in this work, MG does not have any adverse effect when users strictly adhere to the recommended concentration criteria for ornamental fish.

Andiroba (Carapa guianensis Aubl) is an Amazonian plant whose oil has been widely used in traditional medicine for various purposes, including anti-inflammation. Research reports indicate that the oil can confer antitumor activity due to the presence of fatty acids, which can directly influence cell death mechanisms. Thus, andiroba oil (AO) has gained interest for its potential to be used in antineoplastic therapies. Here, we report an in vitro analysis of the cytotoxic and mutagenic potential of AO in the gastric cancer cell line, ACP02. Cell survival was assessed by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, differential staining with ethidium bromide and acridine orange assessed apoptosis-necrosis, and mutagenesis was assessed by the micronucleus test. The apolar oil was first diluted in 0.1% dimethyl sulfoxide (DMSO) and then further diluted to six concentrations (0.01, 0.1, 1, 10 and 100 μg/mL and 1 mg/mL) in RPMI medium. Controls included RPMI alone (negative control) and 0.1% DMSO diluted in medium (vehicle control). The MTT test showed that AO significantly reduced cell viability (P < .05) only when the highest tested concentration was applied for 48 hours. The apoptosis/necrosis test showed that the highest concentration of AO induced cell death by apoptosis at 24 and 48 hours. There was no statistically significant increase in the frequency of micronuclei. The ability of the AO to decrease the viability of ACP02 cells via apoptosis, without exerting mutagenic effects, suggests that the oil could be useful as an alternative therapeutic agent for primary tumors of stomach cancer., (© 2020 John Wiley & Sons, Ltd.)

The genus Makalata is a taxonomically complex group of rodents on which few cytogenetic studies have been performed. Most of the published karyotypes were described based only on conventional chromosome staining. Here, we studied the karyotypes of Makalata from two Brazilian Amazonian states, Amapá and Pará, by Giemsa-staining, G- and C-banding, AgNO3-staining and FISH with 18S rDNA and telomeric sequences probes. We observed 2n = 66/FN = 124 in the Pará state population in Makalata sp; and 2n = 72/FN = 128 in the Amapá state population in M. didelphoides. Multiple chromosome rearrangements may have given rise to these karyotypes, which differ significantly from each other and from those reported in the literature. The chromosomal differences among the described Makalata karyotypes can act as a barrier to gene flow; since they are also associated with geographic barriers (e.g., rivers) and numerous molecular differences, they could be seen as evidence for reproductive isolation of populations from genus Makalata. Our data suggest that the genus is chromosomally diverse and the karyotypes may belong to different species. These karyotypes may prove useful as taxonomic markers for these rodents., Competing Interests: The authors have declared that no competing interests exist.

The hyline tribe Lophyohylini includes 87 species of treefrogs, of which cytogenetics aspects have been studied in less than 20% of them. In order to evaluate the evolution of some of its chromosome characters (NOR position, C-bands, and DAPI/CMA3 bands), we studied the karyotypes of 21 lophyohylines, 16 of them for the first time, and analyzed them in a phylogenetic context. Most species showed similar karyotypes regarding chromosome number (2n = 24) and morphology (FN = 48), excepting Phyllodytes edelmoi and Osteocephalus buckleyi with 2n = 22 (FN = 44) and 2n = 28 (FN = 50), respectively. The NOR location was variable among species and provided valuable phylogenetic information. This marker was located in pair 11 in all species of Trachycephalus, Itapotihyla langsdorffii, and Nyctimantis arapapa, representing the plesiomorphic condition of Lophyohylini. Besides, other apomorphic states were recovered for the clades comprising N. rugiceps and N. siemersi (NOR in pair 5), and Dryaderces pearsoni, Osteocephalus, and Osteopilus (NOR in pair 9). Phyllodytes presented variation for NORs position; they were in pair 2 in P. edelmoi, pair 7 in P. melanomystax, and pair 8 in P. gyrinaethes and P. praeceptor. Polymorphisms in size, number, and activity of this marker were observed for N. siemersi, Osteocephalus fuscifacies, and some species of Trachycephalus. Remarkably, in N. siemersi NORs were detected on a single chromosome in the two specimens studied by this technique, raising the question of how this complex polymorphism is maintained. Interstitial telomeric sequences were found in P. edelmoi, P. melanomystax, and Osteocephalus buckleyi, and their presence seems to be not related to the chromosome reorganization events. Finally, some species showed spontaneous rearrangements, possibly as a consequence of an uncommon phenomenon in anuran cytogenetics: the presence of fragile sites or secondary constrictions not associated with NORs. We propose that this rare feature would have played an important role in the evolution of this group of frogs. From the evidence obtained in this and previous studies, we conclude that Lophyohylini presents a complex chromosome evolution., Competing Interests: The authors have declared that no competing interests exist.

The wide variation found in the size of eukaryotic genomes is largely related to the accumulation of repetitive sequences. Studies show that these sequences can go through an evolutionary process (molecular co-optation) and acquire new genomic functions. Cytogenetic studies reveal a wide karyotypic variation between chelonians (order Testudines) (2n = 26-68), attributed mainly to the number of microchromosomes. The study of repetitive DNAs has the potential to provide data on the dynamics of these sequences, and how they influence the organization of the genome. Here, we reveal the first in situ mapping data of 45S rDNA, histone H3 genes, and telomeric sequences, for a species of the genus Rhinoclemmys, R. punctularia. The karyotype described here for R. punctularia is different from previous reports for the diploid complement of this species, with differences probably attributable to centric fissions and pericentric inversions or centromere repositioning. The 45S rDNA are on a single chromosome pair (like in other turtles), telomeric sequences are in terminal position on all the chromosomes, and histone H3 is dispersed in low copy number, with clusters in pericentromeric regions of three chromosome pairs. We report on the presence of a Gypsy retrotransposon insert located within H3 histone of R. punctularia, and the H3 region sequenced contained the open reading frame of the histone sequence. Comparative modeling revealed a functional pattern for the protein, thus suggesting that the Gypsy element might have been recruited for new functions in the genome of this species., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Eukaryotic genomes exhibit substantial accumulation of repetitive DNA sequences. These sequences can participate in chromosomal reorganization events and undergo molecular cooption to interfere with the function and evolution of genomes. In turtles, repetitive DNA sequences appear to be accumulated at probable break points and may participate in events such as non-homologous recombination and chromosomal rearrangements. In this study, repeated sequences of 5S rDNA, U2 snRNA and Tc1/Mariner transposons were amplified from the genomes of the turtles, Podocnemis expansa and Podocnemis unifilis , and mapped by fluorescence in situ hybridization. Our data confirm the 2n=28 chromosomes for these species (the second lowest 2n in the order Testudines). We observe high conservation of the co-located 5S rDNA and U2 snRNA genes on a small chromosome pair (pair 13), and surmise that this represents the ancestral condition. Our analysis reveals a wide distribution of the Tc1/Mariner transposons and we discuss how the mobility of these transposons can act on karyotypic reorganization events (contributing to the 2n decrease of those species). Our data add new information for the order Testudines and provide important insights into the dynamics and organization of these sequences in the chelonian genomes., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Comparative chromosome-painting analysis among highly rearranged karyotypes of Sigmodontinae rodents (Rodentia, Cricetidae) detects conserved syntenic blocks, which are proposed as chromosomal signatures and can be used as phylogenetic markers. In the Akodontini tribe, the molecular topology (Cytb and/or IRBP) shows five low-supported clades (divisions: " Akodon ", " Bibimys ", " Blarinomys ", " Oxymycterus ", and " Scapteromys ") within two high-supported major clades (clade A: " Akodon ", " Bibimys ", and " Oxymycterus "; clade B: " Blarinomys " and " Scapteromys "). Here, we examine the chromosomal signatures of the Akodontini tribe by using Hylaeamys megacephalus (HME) probes to study the karyotypes of Oxymycterus amazonicus (2n = 54, FN = 64) and Blarinomys breviceps (2n = 28, FN = 50), and compare these data with those from other taxa investigated using the same set of probes. We strategically employ the chromosomal signatures to elucidate phylogenetic relationships among the Akodontini. When we follow the evolution of chromosomal signature states, we find that the cytogenetic data corroborate the current molecular relationships in clade A nodes. We discuss the distinct events that caused karyotypic variability in the Oxymycterus and Blarinomys genera. In addition, we propose that Blarinomys may constitute a species complex, and that the taxonomy should be revised to better delimit the geographical boundaries and their taxonomic status.

Libidibia ferrea (juca) is a plant belonging to the Fabaceae (Leguminosae) family, whose antioxidant activity has been widely described in the literature. We evaluated this parameter of Aqueous ethanol extract (AE), ethyl acetate (ACO), chloroform (CLO) and hexane (HEX) extracts of L. ferrea. We then tested the most active extract for its toxicity and ability to inhibit migratory activity in the ACP02 gastric adenocarcinoma cell line in vitro. The AE and ACO extracts both had antioxidant activity, the AE extract showing greater potential. This may reflect that both extracts contained phenolic compounds. Although AE extract showed no cytotoxic, mutagenic or genotoxic effect, it altered cell morphology and migration activity. Analysis of apoptosis/necrosis indicated that this parameter does not appear to account for the apparent ability of AE to inhibit cancer cell migration. We speculate that the morphological changes in AE-treated cells could be due to cytoskeleton alterations related to the presence of myo-inositol in AE extract. Together, our results demonstrate this extract of L. ferrea can act as an exogenous antioxidant and might prove useful in efforts to fight secondary tumors., Competing Interests: The authors have declared that no competing interests exist.

Background: The Neacomys genus is predominantly found in the Amazon region, and belongs to the most diverse tribe of the Sigmodontinae subfamily (Rodentia, Cricetidae, Oryzomyini). The systematics of this genus and questions about its diversity and range have been investigated by morphological, molecular (Cytb and COI sequences) and karyotype analysis (classic cytogenetics and chromosome painting), which have revealed candidate species and new distribution areas. Here we analyzed four species of Neacomys by chromosome painting with Hylaeamys megacephalus (HME) whole-chromosome probes, and compared the results with two previously studied Neacomys species and with other taxa from Oryzomyini and Akodontini tribes that have been hybridized with HME probes. Maximum Parsimony (MP) analyses were performed with the PAUP and T.N.T. software packages, using a non-additive (unordered) multi-state character matrix, based on chromosomal morphology, number and syntenic blocks. We also compared the chromosomal phylogeny obtained in this study with molecular topologies (Cytb and COI) that included eastern Amazonian species of Neacomys, to define the phylogenetic relationships of these taxa., Results: The comparative chromosome painting analysis of the seven karyotypes of the six species of Neacomys shows that their diversity is due to 17 fusion/fission events and one translocation, pericentric inversions in four syntenic blocks, and constitutive heterochromatin (CH) amplification/deletion of six syntenic autosomal blocks plus the X chromosome. The chromosomal phylogeny is consistent with the molecular relationships of species of Neacomys. We describe new karyotypes and expand the distribution area for species from eastern Amazonia and detect complex rearrangements by chromosome painting among the karyotypes., Conclusions: Our phylogeny reflects the molecular relationships of the Akodontini and Oryzomyini taxa and supports the monophyly of Neacomys. This work presents new insights about the chromosomal evolution of this group, and we conclude that the karyotypic divergence is in accord with phylogenetic relationships.

Crenicichla (Cichliformes, Cichlidae) present a highly conserved diploid number 2n=48 with fundamental numbers varying between 52 and 62. We analyzed four species in order to investigate the role of repetitive DNA in chromosome evolution in the genus. Crenicichla johanna , Crenicichla cf. saxatilis and Crenicichla cf. regani have 2n=48 (8 m/sm and 40st/a) and FN=56, while Crenicichla sp. 'Xingu I' has 2n=48 (48 st/a) and FN=48. Different patterns of constitutive heterochromatin distribution were observed including pericentric, interstitial and whole arm C bands. A single chromosome bears 18S rDNA clusters in most species, except C. johanna , where population variation exists in terms of the quantity and distribution of clusters and their association with interstitial telomeric sequences. All species showed hybridization of 5S rDNA sequences in an interstitial region on an acrocentric chromosome pair. The karyotypic differences and maintenance of the diploid number supports chromosome evolution mediated by inversions in Crenicichla The telomeric and 18S rDNA sequence association in various chromosomes of C. johanna are proposed to represent hotspots for breakage, favoring intra-chromosomal rearrangements. The results suggest that repetitive sequences can contribute to microstructural cytogenetic diversity in Crenicichla ., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Achiasmatic male meiosis in scorpions is characterized by a high frequency of gaps, asynaptic regions and multivalent associations. Here, we performed an immunocytogenetic analysis to investigate recombination, and synapsis and chromatin-remodeling events during meiosis of the scorpion Tityus silvestris Our results demonstrate that the synaptonemal complex (SC) begins its organization in the zygotene stage and persists until metaphase I. The advancement of the synaptic process is related to the epigenetic modification histone H3 lysine 27 trimethylation (H3K27m3). The distribution and dynamics patterns of variant γH2AX and recombinase Rad51 during achiasmatic meiosis suggests formation and repair of DNA double-strand breaks (DSBs) during early stages of prophase I. The epigenetic modifications, histone H3 lysine 4 trimethylation (H3K4m3) and histone H3 lysine 9 acetylation (H3K9ac), showed a dispersed distribution along the bivalents, suggesting that transcriptional activity is maintained constitutively during prophase I. However, H3K9ac modifications are absent in constitutive heterochromatin carrying the 45S rDNA in pachytene and post-pachytene stages. Collectively, our data demonstrate that T. silvestris exhibits adaptations to the achiasmatic mode, and suggest that epigenetic modifications may act in the regulation of these mechanisms to favor the normal continuation of meiosis in this scorpion., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Background: The Micronycterinae form a subfamily of leaf-nosed bats (Phyllostomidae) that contains the genera Lampronycteris Sanborn, 1949, and Micronycteris Gray, 1866 (stricto sensu), and is characterized by marked karyotypic variability and discrepancies in the phylogenetic relationships suggested by the molecular versus morphological data. In the present study, we investigated the chromosomal evolution of the Micronycterinae using classical cytogenetics and multidirectional chromosome painting with whole-chromosomes probes of Phyllostomus hastatus and Carollia brevicauda. Our goal was to perform comparative chromosome mapping between the genera of this subfamily and explore the potential for using chromosomal rearrangements as phylogenetic markers., Results: The Micronycterinae exhibit great inter- and intraspecific karyotype diversity, with large blocks of telomere-like sequences inserted within or adjacent to constitutive heterochromatin regions. The phylogenetic results generated from our chromosomal data revealed that the Micronycterinae hold a basal position in the phylogenetic tree of the Phyllostomidae. Molecular cytogenetic data confirmed that there is a low degree of karyotype similarity between Lampronycteris and Micronycteris specimens analyzed, indicating an absence of synapomorphic associations in Micronycterinae., Conclusions: We herein confirm that karyotypic variability is present in subfamily Micronycterinae. We further report intraspecific variation and describe a new cytotype in M. megalotis. The cytogenetic data show that this group typically has large blocks of interstitial telomeric sequences that do not appear to be correlated with chromosomal rearrangement events. Phylogenetic analysis using chromosome data recovered the basal position for Micronycterinae, but did not demonstrate that it is a monophyletic lineage, due to the absence of common chromosomal synapomorphy between the genera. These findings may be related to an increase in the rate of chromosomal evolution during the time period that separates Lampronycteris from Micronycteris.

The taxonomic identification of Lonchothrix emiliae (Rodentia, Echimyidae, Eumysopinae) is problematic because of the overlap of morphological characters with its sister clade represented by species in the genus Mesomys which, like L. emiliae, is distributed throughout the Amazonian biome. Cytogenetic studies reported the karyotype of L. emiliae as 2n = 60/FN = 116, but this karyotype and samples were later designated as M. hispidus. To evaluate the karyotype diversity of Lonchothrix and Mesomys, and to provide data useful as karyological diagnostic characters, in the present study we made a comparative analysis of specimens of L. emiliae and M. stimulax collected from two Brazilian Amazonian localities, using C-banding, G-banding, FISH using rDNA 45S and telomeric probes, and Cytochrome-b (Cytb) sequences. The results indicate that L. emiliae has 2n = 64♀, 65♂/FN = 124 and a multiple sexual system (XX/XY1Y2), while M. stimulax has 2n = 60/FN = 116. The Neo-X system found in L. emiliae also occurs in two Proechimys species, but cytogenetic analysis indicated an independent origin for these systems. The rDNA 45S analysis showed interstitial signals at one autosomal pair for each species, while an ITS found in L. emiliae was not coincident with the NOR. The molecular analysis confirmed Lonchothrix and Mesomys are sister genera, and the high level of intraspecific genetic divergence (7.1%) in M. stimulax suggests that it may be a species complex., Competing Interests: The authors have declared that no competing interests exist.

Multiple sex chromosome systems have been described for several mammalian orders, with different species from the same genus sharing the same system (e.g., X 1 X 2 Y or XY 1 Y 2 ). This is important because the translocated autosome may be influenced by the evolution of the recipient sex chromosome, and this may be related to speciation. It is often thought that the translocation of an autosome to a sex chromosome may share a common origin among phylogenetically related species. However, the neo-X chromosomes of Proechimys goeldii (2n = 24♀, 25♂/NFa = 42) and Proechimys gr. goeldii (2n = 16♀, 17♂/NFa = 14) have distinct sizes and morphologies that have made it difficult to determine whether they have the same or different origins. This study investigates the origins of the XY 1 Y 2 sex chromosome determination system in P. goeldii (PGO) and P. gr. goeldii (PGG) and elucidates the chromosomal rearrangements in this low-diploid-number group of Proechimys species. Toward this end, we produced whole-chromosome probes for P. roberti (PRO; 2n = 30♂/NFa = 54) and P. goeldii (2n = 25♂/NFa = 42) and used them in comparative chromosomal mapping. Our analysis reveals that multiple translocations and inversions are responsible for the karyotype diversity of these species, with only three whole-chromosomes conserved between PRO and PGO and eight between PGO and PGG. Our data indicate that multiple sex chromosome systems have originated twice in Proechimys. As small populations are prone to the fixation of chromosomal rearrangements, we speculate that biological features of Rodentia contribute to this fixation. We also highlight the potential of these rodents as a model for studying sex chromosome evolution.

Glyphorynchus spirurus (GSP), also called the Wedge-billed Woodcreeper (Furnariidae) has an extensive distribution in the Americas, including the Atlantic coast of Brazil. Nevertheless, there is no information about its karyotype or genome organization. To contribute to the knowledge of chromosomal evolution in Passeriformes we analysed the karyotype of Glyphorynchus spirurus by classic and molecular cytogenetics methods. We show that Glyphorynchus spirurus has a 2n = 80 karyotype with a fundamental number (FN) of 84, similar to the avian putative ancestral karyotype (PAK). Glyphorynchus spirurus pair 1 was heteromorphic in the Tapajós population whereby the short arms varied in sizes, possibly due to a pericentric inversion, as described in other Furnariidae birds. FISH with the Histone H5 probe revealed a signal in the pericentromeric region of G. spirurus chromosome 5 and rDNA 18S showed interstitial signal in GSP-1. Chromosome painting with Gallus gallus (GGA) macrochromosomes 1-9 probes showed disruption of chromosome syntenies of GGA-1, 2 and 4 by fission in Glyphorynchus spirurus. Our results confirm that the GGA1 centric fission is a synapomorphic character for the phylogenetic branch composed of Strigiformes, Passeriformes, Columbiformes and Falconiformes. On the other hand, the GGA-2 fission is reported here for the first time in Passeriformes. Chromosome painting with BOE whole chromosome probes confirmed these rearrangements in Glyphorynchus spirurus revealed by Gallus gallus 1-9 probes, in addition to enabling the establishment of genome-wide homology map., Competing Interests: The authors have declared that no competing interests exist.

Microsatellites show great abundance in eukaryotic genomes, although distinct chromosomal distribution patterns might be observed, from small dispersed signals to strong clustered motifs. In Neotropical fishes, the chromosome mapping of distinct microsatellites was employed several times to uncover the origin and evolution of sex and supernumerary chromosomes, whereas a detailed comparative analysis considering different motifs at the chromosomal level is scarce. Here, we report the chromosomal location of several simple sequence repeats (SSRs) in distinct electric knife fishes showing variable diploid chromosome numbers to unveil the structural organization of several microsatellite motifs in distinct Gymnotus species. Our results showed that some SSRs are scattered throughout the genomes, whereas others are particularly clustered displaying intense genomic compartmentalization. Interestingly, the motifs CA, GA, and GAG exhibited a band-like pattern of hybridization, useful for the identification of homologous chromosomes. Finally, the colocalization of SSRs with multigene families is probably related to the association of microsatellites with gene spacers in this case.

Loricariidae is a diverse group of fish from the neotropical region, occupying a wide variety of freshwater environments. Cytogenetic data have brought important insights into Loricariidae diversity because they help validate undescribed species as well as our understanding of inter- and intraspecific diversity. However, conventional cytogenetic approaches are limited in their ability to detect variability in some lineages, as seen in the Peckoltia clade, owing to their apparent conserved karyotype. Thus, the aim of this work was to map 5S and 18S ribosomal (rDNA) sites in five species of Peckoltia and one species of Ancistomus from the Amazon basin, and discusses the mechanisms of organization and diversification of these clusters. The species analyzed were found to have 2n = 52 and share KF = 38 m-sm +14st-a chromosomes, except Peckoltia vittata with KF = 34 m-sm +18st-a. Extensive variations in the number and location of 5S and 18S rDNA sites were observed among species. These data indicate that inversions are not the most important events in karyotype evolution in this group, and should prove useful in identifying the species studied here. In addition to inversions, transpositions are important evolutionary events that are involved at least in rDNA clusters spreading in Peckoltia and probably in other species of Hypostominae.

Cytogenetic studies show that there is great karyotypic diversity in order Testudines (2n = 26-68), and that this may be mainly attributed to the presence/absence of microchromosomes. Members of the Podocnemididae family have the smallest diploid numbers of this order (2n = 26-28), which may be a derived condition of the group. Diverse studies suggest that repetitive-DNA-rich sites generally act as hotspots for double-strand breaks and chromosomal reorganization. In this context, we used fluorescent in situ hybridization (FISH) to map telomeric sequences (TTAGGG)n, 45S rDNA, and the genes encoding histones H1 and H3 in two species of genus Podocnemis. We also observed conservation of the 45S rDNA and H1 histone sequences (probable case of conserved synteny), but multiple conserved and non-conserved clusters of H3 genes, which colocalized with the interstitial telomeric sequences in the Podocnemis genome. Our results suggest that fusions have occurred between macro and microchromosomes or between microchromosomes, leading to the observed reduction in diploid number in the family Podocnemididae., Competing Interests: The authors have declared that no competing interests exist.