Two different distribution patterns for 5S rDNA sites were revealed by FISH in the four species of grasshoppers. In the first pattern, the site is restricted to a single pair of chromosomes, and in the second pattern, two sites are located in different chromosome pairs. We found six sites in all, including three located in the fourth chromosome and the three in the seventh chromosome, in the order of decreasing size. One or two chromosome pairs with the 5S rRNA gene were reported in 29 grasshopper species from the Acrididae family . Of these 29 species, 13 showed multiple 5S rDNA sites located in the fourth or seventh pairs of chromosomes or even in both pairs (fourth and seventh). In addition, four representatives of the family Proscopiidae, which is composed of the most primitive grasshoppers, showed the 5S rDNA gene located only in the fourth autosomal pair . The 5S rDNA in the fourth pair is also present in B. gigas, C. speciosa, and X. robustus, supported by the results from the Acrididae and Proscopiidae families [16, 24], which suggests that this may be an ancient placement for these genes. C. nuptialis probably lost the site in the fourth pair in the course of evolution.
The 5S rDNA gene observed in the seventh chromosome pair may be an apomorphic condition in grasshopper species in this study, and it may be caused by specific spread mechanisms that remain unclear. Our data also shows all sites located in the proximal region of a medium-sized chromosome, suggesting that this is an ancestral condition for Romaleidae grasshoppers. Despite the spread of this sequence through the Romaleidae genome, the cluster location of the 5S rDNA gene has been restricted to this chromosomal group. We presume that the proximal location of the 5S rDNA cluster is the optimal condition in grasshoppers and probably the ancient one; the other patterns may represent a derived condition. This understanding is also supported by the proximal location of 5S rDNA clusters found in 75% of 29 species of Acrididae and three species of Proscopiidae grasshoppers analyzed [16, 24].
Data on the 18S rDNA gene are scarce in the Romaleidae family, as well as in Ommexechidae and Proscopiidae. Data may be most plentiful for the Acrididae family [10, 15, 16, 24]. The 18S rDNA clusters are restricted to a single medium-sized autosomal pair in C. nuptialis, C. speciosa, and X. robustus, corroborating previous studies from Loreto et al.  and Souza et al. , which suggests that it may be the ancestral bearer of 18S rDNA. This hypothesis is supported by the data from X. d. angulatus, another Romaleidae grasshopper, which showed the 18S rDNA gene in a medium-sized pair . In a study of 47 species of Acrididae and two species of Romaleidae, Cabrero and Camacho  observed that in 32.7% of the species the cluster of 45S rDNA was in a single pair, in 37.7% the site was located in two chromosomes, and in 30.6% the 45S rDNA cluster was in multiple pairs of chromosomes. These data show the 45S rDNA spread through the grasshoppers’ genome in the course of evolution. Two species of Ommexechidae, Ommexecha virens and Descampsacris serrulatum had three and four 45S rDNA sites, respectively. These data show a possible spread of this cluster in these two species . However, in four Proscopiidae grasshoppers there was a consensus location in the seventh chromosome pair, indicating an ancestral condition with this single pair as the original bearer of the 18S rDNA cluster and the extra sites present in two species as a derived pattern .
As a number of species have only a single site for 45S rDNA,, there may be some restrictions on high numbers of rDNA loci within species. The spread can be explained by chromosome rearrangements, translocations or inversions, ectopic recombination, and transposition of some rDNA sequences to new places in the same or different chromosomes . In grasshoppers, the rDNA is sometimes located in constitutive heterochromatin (CH) regions, so a possible rDNA spread mechanism may be associated with CH spread. Previous studies have reported an amplification and dispersion of CH in eight species from six genera (Brasilacris, Chromacris, Phaeoparia, Radacridium, Xestotrachelus, and Xyleus) from the Romaleidae family [21, 23, 25–27]. On the other hand, when data from the 10 ribosomal sites found in B. gigas, C. nuptialis, C. speciosa, and X. robustus are correlated with information from previous studies [22, 25] related to the CH pattern, the results indicate four ribosomal sites in CH regions and six ribosomal sites outside CH regions.
In our study, due to the single site for rDNA in the four species studied, we can infer two important aspects of the location of the nucleolus organizer regions (NORs) in this group: i) The distribution of NORs seems to be well conserved and there are few rearrangements of location and expression of these sequences, and ii) the presence of only one NOR is sufficient to provide the cellular processes in these species. In the megameric chromosome M9 from B. gigas, there is a conspicuous CH block , which encompasses both the 18S rDNA and H3 histone genes, and this arrangement may be associated with a rearrangement leading to a contiguous configuration of these genes. The difference in chromosomal location of the 18S rDNA gene between the two Chromacris species is probably due to a paracentric inversion, an unequal crossover, or even a heterochromatin amplification that led to this modification.
As observed in humans and plants, the rDNA multigene family does not have a random distribution in karyotypes with acrocentric chromosomes [28–30]. This condition was observed in the 5S and 18S rDNA distribution pattern in grasshoppers with acrocentric chromosomes from different families (Acrididae, Ommexechidae, Proscopiidae, and Romaleidae). This pattern found in representatives of Romaleidae might suggest that the ancestral chromosome location for this group is near the centromere. In support of this idea, studies have reported the proximal chromosomal location of active NORs in some Romaleidae grasshoppers, including Radacridium mariajoseae, X. d. angulatus, and Phaeoparia megacephala, [21, 26, 27]. In 47 Old World grasshoppers, the 126 rDNA sites detected by FISH showed that 52.4% were located in the proximal region, 34.9% were interstitial, and only 12.7% were distal . The majority of sites mapped to date in grasshoppers were located in the proximal or pericentromeric regions in acrocentric chromosomes [10, 15, 16].
In the four species of Romaleidae analyzed in our study, we did not observe an association between the two 5S and 18S rDNA markers. They were located in different chromosome pairs. This finding suggests that there are separate evolutionary pathways for these two multigene families in the Romaleidae grasshoppers. This condition is predominant among eukaryotes and was also observed in other grasshoppers from the Acrididae and Proscopiidae families. However, extreme cases have been reported in one grasshopper species Omocestus bolivari, which carries both rDNA types in all its chromosomes [16, 24].
The H3 histone gene has been mapped in 35 representatives from Acrididae and four representatives from Proscopiidae. In all cases, the H3 histone gene cluster was restricted to a single autosomal pair [16, 18]. Herein, the H3 histone gene distribution pattern is described for the first time for Romaleidae grasshoppers. We found that the location of the H3 gene was highly conservative in the four species, with slight modifications concerning location within the chromosome. In all species, the H3 histone gene site is restricted to a single autosome pair. For Chromacris species and X. robustus, the H3 cluster was located in the L2 chromosome, and for B. gigas, it was in the megameric M9. The chromosomal location of the H3 gene cluster was proximal in B. gigas and C. speciosa and terminal in C. nuptialis and X. robustus. The differences in the location of the H3 gene between the two Chromacris species are probably due to chromosomal rearrangements or transposition.
Due to morphologic similarities like wing patterns and phallic structures between the genera Chromacris and Xestotrachelus, Roberts and Carbonell  report in a revision that these two genera are close. Our cytogenetic study showed a similar pattern among the species from the two genera and corroborates the taxonomic relationships among them.
Double FISH revealed the 18S rDNA and H3 histone genes in the same megameric chromosome in B. gigas, indicating a contiguous arrangement of these two genes in the proximal region of the M9 chromosome. According to a study of active NORs by Rufas et al. , megameric and sexual chromosomes are frequently involved in nucleolus organization. Megameric chromosomes show consistent positive heteropycnosis at first prophase in meiosis in orthopteran insects. Rufas et al. showed that a high proportion of Acrididae species carry active NORs in the sex and megameric chromosomes. However, no megameric chromosomes were known to carry H3 histone genes until now. Nine species of Acrididae grasshoppers showed one chromosome pair carrying rDNA-H3 histone genes, but none of the sites was present in the megameric chromosomes [10, 18].
The H3 histone gene pattern found in B. gigas is very different from that of other Romaleidae grasshoppers. This difference is probably due to a modification of the site location from its original place in the L2 to the M9 chromosome. If there is some evolutionary mechanism that combines repetitive elements of rDNA and histone in a megameric chromosome, we do not know what it is. Further chromosomal studies must be done exploring the mobility of rDNA and the highly conservative nature of the H3 histone gene.