Short Communication |
Corresponding author: Renata da Rosa ( renata-darosa@uel.br ) Academic editor: Christina Nokkala
© 2020 Jaqueline Fernanda Dionísio, Joana Neres da Cruz Baldissera, Angélica Nunes Tiepo, José Antônio Marin Fernandes, Daniel Ricardo Sosa-Gómez, 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:
Dionísio JF, da Cruz Baldissera JN, Tiepo AN, Fernandes JAM, Sosa-Gómez DR, da Rosa R (2020) New cytogenetic data for three species of Pentatomidae (Heteroptera): Dichelops melacanthus (Dallas, 1851), Loxa viridis (Palisot de Beauvois, 1805), and Edessa collaris (Dallas, 1851). Comparative Cytogenetics 14(4): 577-588. https://doi.org/10.3897/CompCytogen.v14.i4.56743
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In this paper, we present new cytogenetic data for three species of the family Pentatomidae: Dichelops melacanthus (Dallas, 1851), Loxa viridis (Palisot de Beauvois, 1805), and Edessa collaris (Dallas, 1851). All studied species presented holocentric chromosomes and inverted meiosis for the sex chromosomes. D. melacanthus has 2n = 12 (10A + XY); L. viridis showed 2n = 14 (12A + XY); and E. collaris showed 2n = 14 (12A + XY). C-banding was performed for the first time in these species and revealed terminal and interstitial heterochromatic regions on the autosomes; DAPI/CMA3 staining showed different fluorescent patterns. In all species, fluorescence in situ hybridization (FISH) with 18S rDNA probe identified signals on one autosomal bivalent, this being the first report of FISH application in the species D. melacanthus and L. viridis. The results obtained add to those already existing in the literature, enabling a better understanding of the meiotic behavior of these insects.
Heterochromatin, Holocentric chromosome, Meiosis, Pentatomidae, rDNA-FISH
The suborder Heteroptera has approximately 40,000 species distributed in seven infraorders (Enicocephalomorpha, Dipsocoromorpha, Gerromorpha, Nepomorpha, Leptopodomorpha, Cimicomorpha, and Pentatomomorpha) and is considered the largest and most diverse group of hemimetabolous insects (
Pentatomidae are considered the fourth largest family in the suborder Heteroptera, with approximately 900 genera and almost 4,800 species classified in 10 subfamilies with a worldwide distribution (
Several cytogenetic studies have been conducted on this insect family, where more than 300 species have been analyzed (
Because of the importance and diversity of the family Pentatomidae, we present cytogenetic data for three species of Pentatomidae in this paper: Dichelops melacanthus (Dallas, 1851), Loxa viridis (Palisot de Beauvois, 1805), and Edessa collaris (Dallas, 1851).
For this study, only male adults were used (Table
Species | Number of samples (N) | Collection site |
---|---|---|
Dichelops melacanthus | 40 | District of Maravilha, Londrina, Paraná, Brazil (23°28'03"S, 51°00'46.3"W) |
Loxa viridis | 15 | Iguaçu National Park in Foz do Iguaçu, Paraná, Brazil (25°04'–25°41"S, 53°58'–25°04"W) |
Edessa collaris | 15 | Iguaçu National Park in Foz do Iguaçu, Paraná, Brazil (25°04'–25°41"S, 53°58'–25°04"W) |
The slides were submitted to C-banding following the protocol of
Total DNA was extracted using the phenol-chloroform method of Sambrook and Russel (2006). The 18S rDNA probe was obtained via a polymerase chain reaction (PCR) using the primers Forward 5'-CCTGAGAAACGGCTACCACATC-3' and Reverse 5'-GAGTCTCGTTCGTTATCGGA-3', as described by
FISH was based on the protocol of
The slides were analyzed in an epifluorescence microscope (Leica DM 2000), which was equipped with a digital camera Moticam Pro 282B. The images were captured using Motic Images Advanced software, version 3.2. The chromosome images were acquired separately with specific filters for each fluorophore or in light field.
The stink bug D. melacanthus had 2n = 12 (10A + XY) (Fig.
In all analyzed species, a heterochromatic region corresponding to sex chromosomes was observed, which are associated in the early stages of meiosis (Fig.
FISH revealed the following distribution patterns of 18S rDNA among species: D. melacanthus showed discrete dots in the terminal region of the larger bivalent at metaphase I (Fig.
Meiotic stages of Dichelops melacanthus (a–f), Loxa viridis (g–l) and Edessa collaris (m–r). (a, g and m) metaphase I by Giemsa conventional staining; (b, h and n) metaphase II by Giemsa conventional staining; (c, i and o) C-banding pachytenes; (d and j) metaphase I by DAPI staining; (p) diplotene by DAPI staining; (e and k) metaphase I by CMA3 staining; (q) diplotene by CMA3 staining; (f, l and r) Fluorescence in situ hybridization with digoxigenin-labeled 18S rDNA probe and counterstained with DAPI. X and Y correspond to the sex chromosomes. Arrows show heterochromatic marks in autosomes. Scale bar: 10 µm.
Conventional staining analysis performed here confirmed the presence of holocentric chromosomes and kinetic activity localized in the terminal region during meiosis, as observed in most Heteroptera (
According to data available for Pentatomidae (
The stink bug D. melacanthus was the only species in the study that presented 2n = 12 (10A + XY), a result confirming previous observations reported for other populations of this species (
Apart from the differences in the diploid number, D. melacanthus was distinguished by the presence of a pair of autosomal chromosomes of large size in relation to the other chromosomes. According to
In all species studied, the location and composition of heterochromatin was first performed. In relation to the characteristics of heterochromatin in the autosomes, we can classify the species studied into two distinct patterns: (i) presence of AT-rich heterochromatin as in D. melacanthus and (ii) predominance of DAPI+ blocks and few CMA3+ blocks as in L. viridis and E. collaris. According to
The heterogeneity of heterochromatin in chromosomes was observed. In D. melacanthus and L. viridis, the sex Y chromosome was completely heterochromatic and DAPI+, while the sex X chromosome in these two species showed homogeneous staining with both DAPI and CMA3. In most species of Heteroptera, the Y chromosome presents a large amount of heterochromatin, sometimes being completely heterochromatic (
In this study, E. collaris presented associated sex chromosomes and DAPI+/CMA3+ in early meiotic phases. This has also been reported in Nabis viridulus Spinola, 1837 (
Studies on the characterization and localization of heterochromatin are important because in addition to the numerous functions that it performs during the cell cycle, it is related to karyotype evolution since chromosomal breaks and rearrangements occur frequently in these regions (
Signals of 18S rDNA in a single bivalent were observed for all species of this study, and this pattern is commonly found in the species of the Pentatomidae (
In this study, first data on FISH with the 18S rDNA probe with D. melacanthus and L. viridis are presented and both species showed terminal blocks in autosomes, being the larger bivalent in D. melacanthus. This terminal location is highly conserved in the infraorder Pentatomomorpha, even in related species that exhibit wide variations in chromosome number; chromosome position of the 18S rDNA sites is commonly sub-terminal (
Our results confirm the karyotype conservation of the family and present original cytogenetic data for three species: (i) analysis of heterochromatin in all species; and (ii) FISH with 18S rDNA probe data for D. melacanthus and L. viridis. In conclusion, we present new data for future studies that can collaborate in the evolutionary study of the Pentatomidae family.
The authors would like to thank the Embrapa-Soja and Iguaçu National Park for assistance in collecting insects. This paper was suported by Universidade Estadual de Londrina (UEL); Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES – Finance code 001) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).