Chromosome study in Schistocerca ( Orthoptera-Acrididae-Cyrtacanthacridinae ) : Karyotypes and distribution patterns of constitutive heterochromatin and nucleolus organizer regions ( NORs )

Chromosome analyses were performed in two grasshopper species of the genus Schistocerca, S. pallens and S. flavofasciata. Both species shared the same diploid number (2n = 23, X in males; 2n = 24, XX in females);and a conserved karyotype composed exclusively of acrocentric chromosomes, but differed in their distribution patterns of constitutive heterochromatin and nucleolus organizer regions (NORs). Constitutive heterochromatin was located in the pericentromeric region of all chromosomes in both species. S. flavofasciata presented an additional C-band on the distal region of the long arm of a small autosome pair (S9). Nucleolus organizer regions (NORs), revealed by silver nitrate staining (Ag-NORs), were observed on a medium autosome pair (M5) in both species. S. pallens presented an additional NOR-bearing autosome (M6). The same sites were labeled after FISH with an rDNA probe in S. pallens cells.


Introduction
Cyrtacanthacridinae, a subfamily of the Acrididae family, comprises around 30 genera distributed worldwide through tropical and subtropical regions.In America, it is represented by the genera Halmenus and Schistocerca.The latter has a large number of species and subspecies distributed in North, South and Central America.The most accepted hypothesis proposes that Schistocerca reached America from Africa (Dirsh, 1974;Carbonell, 1977).
In this work, the karyotypes of S. pallens and S. flavofasciata were analyzed by conventional staining, C-banding and Ag-NOR.Additionally, in S. pallens was used the CMA 3 /DA/DAPI staining to qualify the constitutive heterochromatin and FISH with a 45S rDNA probe to map the ribosomal sites.

Material and Methods
Chromosome analyses were performed in Schistocerca pallens and S. flavofasciata collected in different areas of the State of Pernambuco, Northeastern Brazil (Table 1).Cytological preparations were obtained through squashing of testes and ovarian follicles.Mitotic embryo cells of S. pallens were obtained from eggs incubated at 30 °C for 13 days (Souza, 1991).Conventional chromosome analyses were performed after staining with 2% lacto acetic orcein.C-banding was obtained according to Sumner (1972) and CMA 3 /DA/DAPI staining was performed according to Schweizer et al. (1983) with small modifications.For sequential staining (AgNO 3 /CMA 3 / DA/DAPI), the chromosomes were stained with silver nitrate, photographed, destained and stained with CMA 3 /DA/ DAPI.Fluorescent in situ hybridization (FISH) was performed according to Moscone et al. (1996).A probe containing a fragment of Arabidopsis thaliana 45S ribosomal genes (18S-5.8S-28S)(Unfried et al.,1989;Unfried and Gruendler 1990) was labelled with biotin-11-dUTP by nick translation (Life Technologies) and detected with TRITC (tetramethyl-rhodamine-isothiocyanate)-conjugated antibodies.The chromosome preparations were counterstained with DAPI and mounted with Vectashield H-100 (Vector).Photographs were taken with Kodak T-MAX 400 and ISO FUJI 400.
C-banding evidenced pericentromeric constitutive heterochromatin (CH) in all chromosomes of both species (Figure 2a-d).S. flavofasciata presented an additional large distal heterochromatic block on pair S9.Mitotic embryo cells of S. pallens, with more distended chromosomes, presented interstitial CH blocks in two autosome pairs (data not shown).
In meiotic cells of S. pallens, CMA 3 staining highlighted the small interstitial CH blocks on bivalents M5 and M6, which are thus GC-rich (Figure 3b,c).An additional interstitial CH CMA 3 -positive block was observed on M4.All chromosomes were uniformly stained by DAPI in that species.Silver nitrate staining (AgNO 3 ) revealed interstitial NORs on a medium-sized bivalent, probably M5, in thirty pachytene cells of S. flavofasciata (Figure 3e).Ag-NORs were interstitially located on bivalents M5 and M6 in 45 pachytene cells of S. pallens (Figure 3a).Fluorescent in situ hybridization (FISH) with a 45S rDNA probe revealed ribosomal sequences in 15 cells analyzed in each of three specimens of S. pallens (Figure 3d).FISH signals on M5 and M6 coincided with Ag-NORs.The sequential staining AgNO 3 /CMA 3 /DA/DAPI showed that the Ag-NOR-associated heterochromatin is CMA 3 -positive (GC-rich) in this species (Figure 3a-c).

Discussion
In spite of th large number of Schistocerca species in the American continent, only four species had their conventional stained karyotypes described so far (Mesa et al., 1982).C-banding and Ag-NOR staining have been used thoroughly in chromosome analyses of Acridoidea (Rufas et al., 1985;Cabrero and Camacho 1986a;Souza and Kido 1995;Rocha et al., 2004), but not in American Schistocerca.In S. gregaria, a very common Old World species, pericentromeric heterochromatic blocks were described on all chromosomes.Pairs L2 and M6 showed distal CH blocks and pair S9 presented a proximal block of low staining intensity (Hagele, 1979).Ag-NOR staining revealed two active NORs on the distal region of M6 and on the proximal region of S9 (Fox and Santos 1985), which coincided with rDNA FISH signals (Vaughn et al., 1999).In the present study, S. pallens and S. flavofasciata showed similar patterns of pericentromeric CH distribution, but differed by the presence of small blocks on three medium- The pericentromeric CH of S. pallens was not entirely stained by the base-specific fluorochromes CMA 3 and DAPI, but small CMA 3 -positive blocks were detected on pairs M4, M5 and M6.This indicates base composition heterogeneity of the heterochromatin, and shows that most CH in this species is not exclusively AT-or GC-rich.Species of taxonomically related Acrididae and Romaleidae (John and King 1983;John et al., 1985;Bella et al., 1993;Rodriguez-Inigo et al., 1993;Loreto and Souza, 2000;Rocha et al., 2004;Souza and Kido 1995;Pereira and Souza 2000;56 Chromosome study in Orthoptera Souza et al., 2003), have very similar karyotypes, but may differ in heterochromatin distribution and base-pair composition.In the acridoids Chorthippus brunneus and C. jacobsi, the pericentromeric regions of all chromosomes showed CMA 3 -positive CH blocks (Bridle et al., 2002).The romaleids Xyleus angulatus, Phaeoparia megacephala and Xestotrachelus robustus (Souza et al., 1998;Pereira and Souza, 2000;Souza et al., 2003) also presented CMA 3positive CH blocks on all chromosomes.Differences in size and distribution of CH blocks have been described in several grasshopper species.Chromacris speciosa had large pericentromeric and telomeric CH blocks (Souza and Kido, 1995).In contrast, C. nuptialis predominantly showed small CH pericentromeric blocks, in addition to terminal CH blocks on pairs G1 and P9 and an interstitial block on P10 (Loreto et. al., 2005).Other grasshoppers of the genera Calliptamus, Oeidipoda, Euchorthippus and Radacridium also differed in their C-banding patterns (Rocha et al., 1997;Santos et al., 1983).
The two species herein studied showed different patterns of NOR distribution.The interstitial location of Ag-NORs in S. flavofasciata and S. pallens (rDNA identified by FISH in this species), in contrast to the distal (M6) and proximal NORs (S9) of Schistocerca gregaria (Fox and Santos, 1985), suggest the possible occurrence of inversions including the rDNA loci in the genus.
Variation in the distribution of rDNA sites and/or Ag-NORs, as we observed in S. flavofasciata and S. pallens, have been found in many insect species pointing to the importance of these patterns as phylogenetic markers in the genus Schistocerca.
Specimens of Schistocerca pallens and S. flavofasciata analyzed, collection places and geographical coordinates.S. pallens and by an intensely stained block at the telomere of S9 in S. flavofasciata.The latter represents a karyotypic marker in S. flavofasciata that allows the distinction between these species.