Corresponding author: Renata da Rosa ( email@example.com )
Academic editor: Alexander Bugrov
© 2017 Mariana Bozina Pine, Raquel Bozini Gallo, Carlos Roberto Maximiano da Silva, Larissa Forim Pezenti, Fernando Campos De Domenico, Vilma Loreto, Renata da Rosa.
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: Bozina Pine MB, Gallo RB, da Silva CRM, Pezenti LF, De Domenico FC, Loreto V, Rosa R (2017) Chromosome mapping in Abracris flavolineata (De Geer, 1773) (Orthoptera) from the Iguaçu National Park – Foz do Iguaçu, Paraná, Brazil. Comparative Cytogenetics 11(2): 203-212. https://doi.org/10.3897/compcytogen.v11i2.10282
In this paper, we present the cytomolecular analysis of a population of Abracris flavolineata collected in the largest fragment of the Brazilian Atlantic forest, the Iguaçu National Park. The diploid number in males was 23 (22+X0), with two large pairs (1–2), 7 medium (3–9), 2 small (10–11) and the X chromosome of medium size. Heterochromatic blocks were evident in the pericentromeric regions of all chromosomes. Heterogeneity in the distribution of heterochromatin was observed, with a predominance of DAPI+ blocks. However, some chromosomes showed CMA3+ blocks and other DAPI+/CMA3+ blocks. The 18S rDNA sites were distributed on the short arms of 5 pairs. In two of these pairs, such sites were in the same chromosome bearing 5S rDNA, and one of the bivalents, they were co-located. Histone H3 genes were found on one bivalent. The results added to the existing cytogenetic studies provided evidence of great karyotypic plasticity in the species. This pliancy may be the result of vicariant events related to the geographical distribution of different populations of A. flavolineata.
Acrididae, Brazilian Atlantic forest, chromosome banding, fluorescence in situ hybridization, grasshopper
The family Acrididae is one of the most speciose, heterogeneous and conceivably the most recent in the Acridoidea group (
Although there are several cytogenetic studies of Neotropical Acrididae species, especially of the subfamily Ommatolampidinae, there are few studies that elucidate the molecular structure of chromosomes. Specific C-banding techniques for the identification and classification of chromosomes have been applied more often in Abracris flavolineata species (De Geer, 1773) (
Knowledge of Ommatolampidinae is limited, considering the diversity of the subfamily. Few cytogenetic tools have been applied, and the only samples that have been analyzed were from southeastern Brazil (Rio Claro/São Paulo state). To improve the knowledge about this species, in particular about this group, it was necessary, not only to use more cytogenetic tools but also study samples from other regions of Brazil. Thus, this work aimed to study specimens of A. flavolineata collected in the Iguaçu National Park (Southern Brazil), a paramount area of the Brazilian Atlantic, using different cytogenetic markers in order to understand the evolutionary mechanisms present in this group of insects.
The current study used twenty male specimens of Abracris flavolineata collected from the Iguaçu National Park, Foz do Iguaçu, Paraná State, Brazil – 25°37'40.67"S; 54°27'45.29"W (DDM). The individuals were identified and deposited in the Museu de Zoologia da Universidade de São Paulo (
In addition to the karyotype studies, genomic DNA was extracted from the muscle tissue of a male specimen using the phenol/chloroform procedure described by Sambrook and Russel (2001). Unlabelled 18S rDNA, 5S rDNA and Histone H3 gene probes were generated by polymerase chain reaction (PCR) using the primers: 18S rDNAF 5‘-CCTG AGAAACGGCTACCACATC-3’ and 18S rDNAR 5‘-GAGTCTCGTTCGTTATCGGA-3’(
All samples of Abracris flavolineata collected in the Iguaçu National Park presented 2n=23 (♂) and an XX/X0 sex-determination system (Fig.
Mitotic and meiotic stages of Abracris flavolineata by Giemsa conventional staining (a–c), C-banding (d–f) and fluorochromes staining (g–i): a mitotic metaphase b diakinesis c metaphase II d pachytene e diakinesis f metaphase II g mitotic metaphase by DAPI staining h mitotic metaphase by CMA3 staining i overlapping DAPI/CMA3. The numbers correspond to autosomes. X corresponds to the sex chromosome. The arrows indicate the discrete heterochromatic bands. Bar= 5µm.
The karyotype structure observed in this study is conserved regarding the diploid number and morphology of the A chromosomes, corroborating the data already established for the species by
Heterochromatic blocks were shown in the pericentromeric regions of all chromosomes (Fig.
Staining with fluorochromes CMA3/DAPI showed heterogeneity in the distribution of heterochromatin on autosomes: (i) DAPI+ bands located at pericentromeric regions in most chromosomes; (ii) pericentromeric DAPI+/CMA3+ bands on 2 and 6 chromosomes; (iii) a discrete CMA3+ band in the terminal region on chromosome 4; and (iv) DAPI+ adjacent to CMA3+ blocks on three pairs (5, 7 and 9). The X chromosome showed adjacency of DAPI+-CMA3+-DAPI+ bands (Figure
There are three patterns of distribution of GC-rich blocks in grasshoppers: (i) CMA3+ bands related to NORs location (
Although the position of rDNA sites was similar to that previously reported for other populations of this species, on terminal region, the number of sites was different from that observed in previous studies. The 18S rDNA sites were distributed on terminal regions of the short arms of all analyzed specimens of 5 pairs (1, 2, 4, 7 and 10), with no variations. Two of these sites were on the same chromosome as 5S rDNA sites. These sites were co-located on one of the bivalents (Fig.
Fluorescent in situ hybridization of A. flavolineata in meiocytes: a, b 18S rDNA (green) and 5S rDNA (red) probes c, d histone H3 gene probe: a pachytene b–d two cells in metaphase II. The numbers correspond to autosomes. X corresponds to the sex chromosome. The arrows indicate the histone H3 gene sites. Bar= 5µm.
Histone H3 genes were located on a corresponding bivalent of pair 8 (Fig.
Idiogram showing the mapping of different sequences studied in Abracris flavolineata from the Iguaçu National Park.
Although the chromosome number and karyotype formula of the specimens studied here were the same as those described in the southeastern population (
The authors are grateful to the National Iguaçu Park staff members for their technical assistance; to Edson Mendes Francisco for his help with the sample collection. This work was supported by Universidade Estadual de Londrina. The researchers received permission from Instituto Chico Mendes de Conservação da Biodiversidade – ICMBio to collect grasshoper specimens.