New cytogenetic data for three species of Pentatomidae (Heteroptera): Dichelops melacanthus (Dallas, 1851), Loxa viridis (Palisot de Beauvois, 1805), and Edessa collaris (Dallas, 1851)

Abstract 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.


Introduction
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 (Štys and Kerzhner 1975;Weirauch and Schuh 2011). Although many of these insects play an important role as indicators of environmental quality (Brown 1997), other species are responsible for significant economic importance as vectors of diseases (Alevi et al. 2015) and agricultural pests (Schaefer and Panizzi 2000).
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 (Rider 2011). The Neotropical region, which includes Brazil, is known for its vast biodiversity (Goldani 2012), where 230 genera and more than 1,400 Pentatomidae species have already been described (Grazia et al. 2015).
Several cytogenetic studies have been conducted on this insect family, where more than 300 species have been analyzed (Ueshima 1979;Rebagliati et al 2005;Kaur and Semahagn 2010;Kerisew 2012;Souza et al. 2007Grozeva et al. 2011;Bardella et al. 2013aBardella et al. , 2016a. The diploid numbers ranging from 2n = 6 in Rhytidolomia senilis (Say, 1832) to 2n = 27 in Thyanta calceata (Say, 1832) with a predominance of 2n = 14 and sex chromosome system XX/XY. These insects like the rest species of Heteroptera have specific cytogenetic features, such as (i) holocentric chromosomes; (ii) kinetic activity located in the terminal region of chromosomes during meiosis (telokinetic activity); (iii) chiasmatic autosomal bivalents, in contrast to the sex chromosomes that are achiasmatic; and (iv) inverted meiosis in the sex chromosomes, which is different from the typical equation reduction-sequence observed in the meiosis of organisms with monocentric chromosomes (Ueshima 1979;Nokkala 1984, 1997;Nokkala and Grozeva 2000;Pérez et al. 2000;Poggio et al. 2009;Melters et al. 2012).
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).

Chromosomal preparations and conventional staining
For this study, only male adults were used (Table 1). Specimens were collected with the authorization of the ICMBio (31946-4). The insects were anesthetized and dissected in a physiological solution for insects (7.5 g NaCl, 2.38 g Na 2 HPO 4 , and 2.72 g KH 2 PO 4 in 1 l of distilled water). The gonads were washed with tap water and fixed in methanol and acetic acid (3:1, v:v). The slides were prepared based on the protocol of Pijnacker and Ferwerda (1984), using a portion of the testes, which was macerated in 45% acetic acid and then dried on a hot plate at 45-50 °C. These preparations were stained using conventional staining with Giemsa 3%.

C-banding and fluorochromes
The slides were submitted to C-banding following the protocol of Sumner (1972) with the modifications of Grozeva et al. (2011). The slides were treated with 0.2 N HCl solution at room temperature for 30 min, incubated in 5% barium hydroxide solution at room temperature for 8 min, and then incubated in 2 × SSC saline at 60 °C for 1 h. The slides were washed with distilled water; some were stained with propidium iodide according to Lui et al. (2012), and others were stained with the fluorochromes 4'6-diamidino-2-phenylindole (DAPI), which identify AT-rich regions, and chromomycin A3 (CMA 3 ), which identify GC-rich regions (Schweizer 1980).

Fluorescence in situ hybridization (FISH)
FISH was based on the protocol of Schwarzacher and Heslop-Harrison (2000): the slides were treated with RNase (0.4% RNAse/2 × SSC) and pepsin (0.005%) for 1 h and 10 min, respectively, both at 37 °C, and dehydrated in ethanol series (75% and 100%) for 3 min each. Subsequently, 40 μl of the hybridization mix containing 100% formamide (20 μl), 50% polyethylene glycol (8 μl), 20 × SSC (4 μl), 10% sodium dodecyl sulfate (SDS) (1 μl), and 100 ng of probes (4 μl) was added to ultra-pure water to complete the reaction. The hybridization mix was denatured at 75 °C for 10 min and then was transferred to the ice. After this time, the mix was applied onto the slides and taken to the thermocycler for denaturing/renaturing following the steps (90 °C, 56 °C, and 38 °C for 10 min each); hybridization occurred at 37 °C in a humidified chamber overnight. After probe detection with anti-digoxigenin-rhodamine (Roche Applied Science, Indianapolis, EUA), the chromosomes were counterstained with DAPI and mounted in Vectashield (Thermo Fisher Scientific, Massachusetts, EUA) and left in the dark for 24 h before analysis. 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.

Results
The stink bug D. melacanthus had 2n = 12 (10A + XY) (Fig. 1a, b) with one pair of autosomes larger than the other autosomes. L. viridis (Fig. 1g, h) and E. collaris (Fig. 1m, n) had chromosomes of homogeneous size and 2n = 14 (12A + XY), in both species. The sex chromosome system of all species was simple (XY male). The analysis of the meiotic behavior revealed a kinetic activity in the terminal regions of the chromosomes owing to their positioning and migration to the opposite poles. In metaphase II, it was possible to observe a radial plate the autosomes forming a ring and the sex chromosomes positioned in the center of the ring in a brief association, known as touch-andgo-pairing in all species (Fig. 1b, h, n).
In all analyzed species, a heterochromatic region corresponding to sex chromosomes was observed, which are associated in the early stages of meiosis (Fig. 1c, i, o). In D. melacanthus, the DAPI/CMA 3 staining was homogeneous for all chromosomes (Fig. 1d, e). In L. viridis presented interstitial and terminal heterochromatic regions in some autosomes (Fig. 1i), and after staining with fluorochromes, the Y chromosome stood out as DAPI + (Fig. 1j). A similar pattern was observed in one of the autosomes but with more discrete coloration; the DAPI + interstitial regions were not so evident in another autosome (Fig. 1j). Staining with CMA 3 showed very weak terminal dots in a bivalent (Fig. 1k). The specie E. collaris showed terminal and interstitial heterochromatic bands in the autosomal bivalents in addition to the sex chromosomes that were associated (Fig. 1o). The DAPI/CMA 3 staining revealed the presence of several interstitial and a terminal DAPI + bands (Fig. 1p) as well as CMA 3 + bright terminal dots in a bivalent, and the DAPI + /CMA 3 + sex chromosomes (Fig. 1p, q). 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. 1f ); two 18S rDNA sites in the terminal region of a bivalent were observed in the initial meiotic phases of L. viridis and E. collaris (Fig. 1l, r).

Discussion
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 (Ueshima 1979;Nokkala 1984, 1997;Nokkala and Grozeva 2000;Pérez et al. 2000;Papeschi et al. 2003;Poggio et al. 2009;Melters et al. 2012). In addition, it was possible to observe the occurrence of inverted meiosis for the sex chromosomes, as evidenced by the presence of these chromosomes as univalents in metaphase I and the presence of heteromorphic chromatids with touch-and-go-pairing behavior in metaphase II, a feature already reported in other species of Pentatomidae (Viera et al. 2009).
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 , the presence of this visibly larger autosomal pair suggests that this reduced karyotype originated through a fusion between two autosomes. The same characteristic has also been observed in other species of the family with 2n = 12, as in E. crassus, D. furcatus, M. maldonadensis, and A. bonariensis (Hughes-Schrader and Schrader 1956;Ueshima 1979;Rebagliati et al. 2001Rebagliati et al. , 2005Rebagliati and Mola 2010), which supports the hypothesis of the fusions resulting in a reduction of the diploid number in this group.
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 CMA 3 + blocks as in L. viridis and E. collaris. According to Poggio et al. (2011), most of the reports concerning the characterization of heterochromatin in the autosomes in species of the order Hemiptera are described as DAPI + , as was reported by Bressa et al. (2005) in Athaumastus haematicus (Stål, 1860), Leptoglossus impictus (Stål, 1859), Phthia picta (Drury, 1770) (Coreidae), Largus rufipennis (Laporte, 1832) (Largidae) and Jadera sanguinolenta (Fabricius, 1775) (Rhopalidae), and by Franco et al. (2006) in Spartocera batatas (Fabricius, 1798) (Coreidae).
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 CMA 3 . In most species of Heteroptera, the Y chromosome presents a large amount of heterochromatin, sometimes being completely heterochromatic (Grozeva and Nokkala 2001). This has been reported for the subfamily Triatominae (Panzera et al. 1995), in species of Belostomatidae (Papeschi 1988) and in three species of pentatomids of the genus Antiteuchus (Dallas, 1851) (Lanzone and Souza 2006). Although studies in Reduviidae show that the DAPI positive Y chromosome is quite common, particularly in species of the Triatomini tribe (Bardella et al. 2016b), it is not observed with the same frequency in Pentatomidae, being pointed out only in Halys serrigera (Westwood, 1837) and Perillus bioculatus (Fabricius, 1775) (Kerisew 2012).
In this study, E. collaris presented associated sex chromosomes and DAPI + /CMA 3 + in early meiotic phases. This has also been reported in Nabis viridulus Spinola, 1837 (Grozeva et al. 2004) and in species of the genus Edessa in E. meditabunda (Fabricius, 1974) and E. rufomarginata (De Geer, 1773) (Rebagliati et al. 2003). 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 (Huisinga et al. 2006;Grewal and Jia 2007). The occurrence of small CMA 3 + blocks and/or dots related to co-localization with the nucleolusorganizing regions is a common feature (Camacho et al. 1985;Rebagliati et al. 2001;Bardella et al. 2013b). We confirmed this for E. collaris, where the heterochromatic dots showed specificity to the fluorochrome CMA 3 , and subsequently by 18S rDNA hybridization. A higher percentage of CG repeats in the nucleotide composition of the 18S gene has already been observed by Bargues et al. (2000) in study with triatomines.
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 Grozeva et al. 2015). Most studies of the Pentatomidae report the presence of this cluster on an autosomal pair Bressa et al. 2008Bressa et al. , 2009Grozeva et al. 2011Grozeva et al. , 2015Bardella et al. 2013aBardella et al. , 2016aSouza-Firmino et al. 2020).
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 subterminal (Bardella et al. 2013a). The species E. collaris showed two signals of hybridization in an autosomal bivalent, as previously reported by Souza-Firmino et al. (2020).
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.