Research Article |
Corresponding author: Diogo C. Cabral-de-Mello ( mellodc@rc.unesp.br ) Academic editor: Vladimir Gokhman
© 2016 Allison Anjos, Vilma Loreto, Diogo C. Cabral-de-Mello.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Anjos A, Loreto V, Cabral-de-Mello DC (2016) Organization of some repetitive DNAs and B chromosomes in the grasshopper Eumastusia koebelei koebelei (Rehn, 1909) (Orthoptera, Acrididae, Leptysminae). Comparative Cytogenetics 10(2): 219-228. https://doi.org/10.3897/CompCytogen.v10i2.7609
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B chromosomes occur in approximately 15% of eukaryotes and are usually heterochromatic and rich in repetitive DNAs. Here we describe characteristics of a B chromosome in the grasshopper Eumastusia koebelei koebelei (Rehn, 1909) through classical cytogenetic methods and mapping of some repetitive DNAs, including multigene families, telomeric repeats and a DNA fraction enriched with repetitive DNAs obtained from DOP-PCR. Eumastusia k. koebelei presented 2n=23, X0 and, in one individual, two copies of the same variant of a B chromosome were noticed, which are associated during meiosis. The C-positive blocks were located in the pericentromeric regions of the standard complement and along the entire length of the B chromosomes. Some G+C-rich heterochromatic blocks were noticed, including conspicuous blocks in the B chromosomes. The mapping of 18S rDNA and U2 snDNA revealed only autosomal clusters, and the telomeric probe hybridized in terminal regions. Finally, the DOP-PCR probe obtained from an individual without a B chromosome revealed signals in the heterochromatic regions, including the entire length of the B chromosome. The possible intraspecific origin of the B chromosomes, due to the shared pool of repetitive DNAs between the A and B chromosomes and the possible consequences of their association are discussed.
Cytogenetics, DOP-PCR, FISH, multigene family, Orthoptera
The grasshoppers of the subfamily Leptysminae (Orthoptera, Acrididae) are divided into two tribes, Leptysmini and Tetrataeniini, comprising 75 species distributed exclusively in the Neotropical region (
B chromosomes are present in approximately 15% of eukaryote species and although discovered in 1907, they remain a mystery regarding their origin and evolution in most species (
The accumulation of repetitive DNAs as an evolutionary process has been frequently reported for B chromosomes (
Ten adult males of E. k. koebelei were collected in Serrolândia/Pernambuco, Brazil. The testes were fixed in Carnoy’s solution (3:1 absolute ethanol:acetic acid) and stored at -20°C until use. For chromosomal preparations, the tissues were macerated in a drop of 50% acetic acid and the slides were dried using a hot plate at 40–45°C. All individuals were studied using conventional staining with 5% Giemsa to describe the general karyotype structure. C-banding was performed according to
The 18S ribosomal DNA (rDNA) sequence and the U2 snDNA were obtained through polymerase chain reaction (PCR) from the genomes of Dichotomius semisquamosus (Curtis, 1845) (Coleoptera, Scarabaeidae) and Abracris flavolineata (De Geer, 1773) (Orthoptera, Acrididae), respectively, using primers described by
The 18S rDNA probe and DOP-PCR product were labeled using biotin-14-dATP through nick translation (Invitrogen, San Diego, CA, USA), while the telomeric probe and U2 snDNA were labeled through PCR with digoxigenin-11-dUTP (Roche, Mannheim, Germany). Fluorescent in situ hybridization (FISH) was performed according to the protocol proposed by
The karyotype of E. k. koebelei is in accordance with previous descriptions (
Conventional staining with Giemsa in meiotic cells of E. k. koebelei harboring B chromosomes. a zygotene b early pachytene c metaphase I d anaphase I e metaphase II. B chromosomes are associated side by side in initial meiosis (a, b) and by centromere in other cells (c–e). These chromosomes are also segregated to the same pole in d and maintained together in metaphase II (e). X and B chromosomes are indicated. Bar: 5 µm.
C-banding revealed pericentromeric C-positive heterochromatic blocks in the A complement (Figure
C-banding (a, d), CMA3 staining (b, e, f) and FISH using as probes DOP-PCR (c, g), 18S rDNA-green and TTAGG-red (h) and U2 snDNA (i) in meiotic cells of E. k. koebelei with B chromosomes (d–i) and without them (a–c). a, h late pachytene b early diakinesis c, d, g diplotene e, i metaphase I f zygotene. Images d–g partially highlight B chromosomes. X, B and other chromosomes harboring specific signals are indicated; arrowheads point to the centromeres of B chromosomes. Inserts in g, h highlight B chromosomes. Bar: 5 µm.
Another argument favoring the notion of repetitive DNA enrichment in the C-positive regions was confirmed through the use of the DOP-PCR fraction as a probe, which revealed strong signals in these areas (Figure
FISH with the telomeric probe revealed terminal signals in all chromosomes, including the B chromosome (Figure
The authors declare that they have no conflict of interest.
This study was supported by Fundação de Amparo a Pesquisa do Estado de São Paulo-FAPESP (process number 2014/11763-8) and Coordenadoria de Aperfeiçoamento de Pessoal de Nível Superior-CAPES. AA acknowledges scholarship obtained from FAPESP (process number 2014/06226-3). DCCM was recipient of a research productivity fellowship from Conselho Nacional de Desenvolvimento Científico e Tecnológico-CNPq (process number 304758/2014-0).