Chromosome mapping of ribosomal genes and histone H4 in the genus Radacridium (Romaleidae)

In this study, two species of Romaleidae grasshoppers, Radacridium mariajoseae and R.nordestinum, were analyzed after CMA3/DA/DAPI sequential staining and fluorescence in situ hybridization (FISH) to determine the location of the 18S and 5S rDNA and histone H4 genes. Both species presented karyotypes composed of 2n = 23, X0 with exclusively acrocentric chromosomes. CMA3+ blocks were detected after CMA3/DA/DAPI staining in only one medium size autosome bivalent and in the X chromosome in R. mariajoseae. On the other hand, all chromosomes, except the L1 bivalent, of R. nordestinum presented CMA3+ blocks. FISH analysis showed that the 18S genes are restricted to the X chromosome in R. mariajoseae, whereas these genes were located in the L2, S9 and S10 autosomes in R. nordestinum. In R. mariajoseae, the 5S rDNA sites were localized in the in L1 and L2 bivalents and in the X chromosome. In R. nordestinum, the 5S genes were located in the L2, L3, M4 and M5 pairs. In both species the histone H4 genes were present in a medium size bivalent. Together, these data evidence a great variability of chromosome markers and show that the 18S and 5S ribosomal genes are dispersed in the Radacridium genome without a significant correlation.


Introduction
The Romaleidae family accounts for more than 200 species of grasshoppers comprising three subfamilies: Romaleinae, Aucacrinae and Trybliophorinae. It is the second most diverse family of the superfamily Acridoidea occurring from semiarid regions to tropical rainforests (Carbonell, 1977;Roberts and Carbonell, 1982;Carbonell, 1984Carbonell, , 1986Carbonell, , 2002. The genus Radacridium is dispersed across the Northeast region of Brazil, where it seems to be well-adapted to the severely arid conditions (Carbonell, 1984). Two species are found in the state of Pernambuco: Radacridium mariajoseae, which is typical of the Agreste region, and Radacridium nordestinum, typical of the Caatinga biome (Carbonell, 1984(Carbonell, , 1996. Cytogenetic studies involving representatives of Romaleidae have revealed a great karyotypic conservation (Mesa et al., 1982(Mesa et al., , 2004Souza and Kido, 1995;Rocha et al., 1997;Pereira and Souza, 2000). Most of analyzed species, including R. mariajoseae and R. nordestinum, presented karyotypes with 2n = 23 male, 24 female, an X0/XX sex chromosome system and exclusively acrocentric chro-mosomes. The constitutive heterochromatin (CH) in Romaleidae is predominantly located in the pericentromeric regions of all chromosomes, as observed in Xyleus angulatus, Brasilacris gigas, Chromacris nuptialis, Radacridium nordestinum e Phaeoparia megacephala (Souza and Kido, 1995;Rocha et al., 1997;Pereira and Souza, 2000). Analyses with DAPI and CMA 3 in romaleids showed a predominance of GC-rich (CMA 3 + ) regions. In Xyleus angulatus and Xestotrachelus robustus, CMA 3 + blocks were observed in all chromosomes (Souza et al., 1998;Souza et al., 2003). In contrast, few GC-rich CH regions were observed by Loreto et al. (2005) in two Chromacris species. C. nuptialis had only one bivalent (M 6 ) with a pericentromeric CMA 3 + block, whereas C. speciosa presented CMA 3 + blocks in two autosomal bivalents, a proximal one in M 6 and a telomeric one in L 2 .
A large part of the eukaryotic genome is formed by repetitive DNA, including tandem sequences mainly comprising satellite DNA and multigene families. Ribosomal DNA and histone gene families include a variable number of copies with various genome locations (Charlesworth et al., 1994;Nei and Rooney, 2005). Fluorescence in situ hybridization (FISH) with rDNA and histone genes probes has proven useful for mapping and their location and in clarifying the genome organization of several organisms. Among invertebrates, rDNA gene mapping has been used in several groups, including worms , mollusks , insects (Cabrero and Camacho, 2008;Cabral-de-Mello et al., 2011a;Panzera et al., 2012), echinoderms (Caradonna et al., 2007) and others. The studies involving histone genes mapping in grasshoppers included some species of the Acrididae family (Cabrero et al., 2009;Oliveira et al., 2011) and four species of the Proscopiidae family (Cabral-de-Mello et al., 2011b) were mapped.
This study aimed at understanding the pattern of organization of multigene families in two species of Radacridium. Our results showed a great variability in the number and location of rDNA clusters in both species, whereas the histone H4 genes were highly conserved in number. These results are compared with data from other grasshopper species and are discussed based on the possible mechanisms involved in repetitive DNA diversification.

Material and Methods
We analyzed ten individuals of R. mariajoseae from Gravatá (08º12'04" S; 35º33'53" W) and Bezerros (08º14'00" S; 35º47'49" W) and ten adult males of Radacridium nordestinum from Surubim (07º49'59" S; 35º45'17" W), all located in the Agreste region of the state of Pernambuco, Brazilian Northeast. Specimens were processed and their testes were fixed in Carnoy solution (3:1 ethanol:acetic acid). Chromosome preparations were obtained by the testicular follicles squashing technique, in one drop of 45% acetic acid. The coverslips were removed after liquid nitrogen immersion.
CMA 3 /DA/DAPI sequential staining was performed according to Schweizer (1976). The slides were stained with CMA 3 (0.5 mg/mL) for one hour, washed with distilled water and stained with DA (Distamicine A, 0.1 mg/mL) for 45 min. The slides were rewashed and stained with DAPI (2 mg/mL) for 20 min and mounted in glycerol/McIlvaine buffer/MgCl 2 .
FISH was performed according to Cabral-de-Mello et al. (2011c), with some modifications. The chromosome preparations were submitted to an alcohol series pre-treatment and then treated with RNase and pepsin. The hybridization mix contained 1 mL of probe and the denaturation was performed in a humid chamber at 75°C, followed renaturation overnight at 37°C. Immunodetection was performed with mouse anti-biotin (M743, DAKO) and rabbit anti-mouse-TRITC (R270, DAKO) for biotin and sheep anti-digoxigenin (Roche 1 207 741) and sheep anti-rabbit-FITC (DAKO-F0135) for digoxigenin. Chromosomes were counterstained with 4,6 diamidine-2-phenyl indole (DAPI) and the slides were mounted with Vectashield (Vector). Images were obtained in a Leica epifluorescence microscope, captured with the CW4000 program (Leica) and adjusted in Adobe Photoshop CS5.

Results
Radacridium mariajoseae and R. nordestinum presented 2n = 23(male), a sex determination mechanism of the X0(male) type and exclusively acrocentric chromosomes. Chromosomes of both species were grouped in three large (L 1 -L 3 ), five medium (M 4 -M 8 ) and three small (S 9 -S 11 ) pairs. The X chromosome was a medium size acrocentric in both species. CMA 3 /DA/DAPI sequential staining showed GC-rich (CMA 3 positive) constitutive heterochromatin (CH) blocks in the interstitial region of only one medium-sized bivalent (M 5 ) and in the pericentromeric region of the X chromosome in R. mariajoseae (Figure 1a). In R. nordestinum, CMA 3 + blocks were present in the interstitial region of the L 2 bivalent and in the pericentromeric region of all chromosomes, except the L 1 bivalent (Figure 1c). DAPI staining was homogeneous in both species (Figure 1b and d).
After FISH, the 18S rDNA probe labeled a single site in the pericentromeric region of the X chromosome in R. mariajoseae ( Figure 2a) and three pericentromeric sites, in the bivalents L 2 , S 9 and S 10 of R. nordestinum (Figure 2b). The 5S rDNA genes were located in the pericentromeric region of the two largest bivalents (L 1 and L 2 ) and in the X chromosome of R. mariajoseae, (Figure 2c) and in the bivalents L 2 , L 3 M 4 and M 5 of R. nordestinumm (Figure 2d). The histone H4 probe was mapped in a proximal location in a medium-sized autosome bivalent (M 5 ) in both species (Figure 2e and f).

Discussion
The karyotypic similarities detected after conventional analysis between the two species of Radacridium did not extend to their CMA 3 and DAPI staining patterns. Our results showed GC-richness CH heterogeneity. In R. mariajoseae only one autosome pair and the X chromosome presented CMA 3 + blocks, which resembled the scarcity in GC-rich regions of other Romaleidae, as two species of Chromacris with a single CMA 3 + block (Loreto et al., 2005). The presence of a large number of CMA 3 + blocks is more frequent in Romaleidae, as observed in Xyleus angulatus, Phaeoparia megacephala and Xestotrachelus robustus, in which all CH was shown to be GC-rich (Souza et al., 1998;Pereira and Souza, 2000;Souza et al., 2003).
A great difference in the 18S and 5S rDNA distribution was observed between the species studied. R. mariajoseae presented a single 18S rDNA site in the X chromosome, confirming the finding of only one active nucleolus organizer region (NOR) after silver nitrate impregnation (AgNO 3 ) by Rocha et al. (1997). Although three autosome bivalents showed 18S hybridization signals in R. nordestinum, only the L 2 pair had a corresponding active NOR identified by Rocha et al. (1997). These data indicate a possible dispersion of the 18S rDNA sequences, which may have been caused by structural chromosome rearrangements, ectopic recombination and transpositions (Cabrero and Camacho, 2008). These kind of events have been observed in other insects (Nguyen et al., 2010;Cabral-de-Mello et al., 2011c). In the genus Radacridium, the ancestral rDNA site could have been present in the M 9 biva-lent in a common ancestor. Two points support this hypothesis: the presence of a 18S rDNA site in this pair in R. nordestinum, which was considered as a megameric chromosome by Rocha et al. (1997) and the preferential localization of NORs is in this type of chromosome in several species of grasshoppers (Rufas et al., 1985). The 18S rDNA probe location in the X chromosome of R. mariajoseae could have resulted from a chromosome rearrangement, such as a translocation or transposition, that moved it from M 9 to the X, as a meiotic association between this chromosome and the megameric one has been described (Loreto et al., 2008a).
18S rDNA sites restricted to autosomes, as observed in R. nordestinum, were also detected in Xestotrachelus robustus, Chromacris nuptialis and C. speciosa (Souza et al., 2003;Loreto et al., 2005). On the other hand, 18S rDNA sites located both in autosomes and in the sex chromosome have been described, for example, in Xyleus discoideus angulatus (Souza et al., 1998;Loreto et al., 338 Chromosome mapping in Radacridium Figure 1 -CMA 3 /DA/DAPI sequential staining in R. mariajoseae (a-b) and R. nordestinum (c-d). In (a) CMA 3 + blocks are only present in the X chromosome and in the M 5 bivalent. In (c) all chromosomes, including the X chromosome, show CMA 3 + blocks, except the L 1 bivalent. DAPI staining was homogeneous for both species (b-d).Scale bar = 10 mm.
2008b). 18S rDNA sites have been frequently observed associated with GC-rich regions in Romaleidae (Pereira and Souza, 2000;Loreto et al., 2005), Acrididae (Loreto and Souza, 2000;Rocha et al., 2004), Proscopiidae (Souza and Moura, 2000) and Ommexechidae (Carvalho et al., 2011), a pattern also observed in the two species analyzed herein. The histone H4 genes were located in a single chromosome pair (M 5 ) in both species. A single chromosome pair bearing histone genes was also described in other species of grasshoppers. Cabrero et al. (2009) analyzed the location of the histone H3 and H4 genes in 35 species of grasshoppers from the Acrididae family. They observed that the great majority of species analyzed showed only one histone site, located in an autosome pair. In the same work, double FISH performed in 11 randomly chosen species revealed that, in all cases, both genes were present in the same chromosome site, indicating a great conservation of histone gene location in Acrididae. Cabral-de-Mello et al. (2011b), using a histone H3 probe in four species of Proscopiidae (Tetanorhynchus silvai, Scleratoscopia protopeirae, S. spinosa and Stiphra robusta), observed a single site in the M4 bivalent in all the species. Oliveira et al. (2011) observed multiples sites of histone H3 in Rhammatocerous brasiliensis (Acrididae) and concluded that this would be a derived condition and the presence of a single histone site, as observed in both species studied herein, would be the ancestral form.
The 5S and the 18S genes co-localized in the L 2 chromosome of R. nordestinum and in the X chromosome of R. mariajoseae. Similarly to what we observed in both Radacridium species, an extensive variation in 5S rDNA distribution has been found in others grasshoppers, in which species presenting single sites and extending to all chromosome pairs have been described (Loreto et al., 2008b;Cabral-de-Mello et al., 2011a).
The results obtained in this study indicate a great level of karyotypic differentiation between R. mariajoseae and R. nordestinum. These data reinforce the fact that the high conservation observed at the chromosome level, including chromosome number and morphology, in Radacridium and other romaleids, is not reflected at the genomic level. Our results also contribute to the understanding of chromosome evolution patterns in the family Romaleidae.