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 , 1984 , 1986 , 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 , 1996 ) .
cytogenetic studies involving representatives of romaleidae have revealed a great karyotypic conservation ( mesa et al . , 1982 , 2004 ; souza 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 chromosomes .
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 . ,
analyses with dapi and cma3 in romaleids showed a predominance of gc - rich ( cma3 ) regions . in xyleus angulatus and xestotrachelus robustus
in contrast , few gc - rich ch regions were observed by loreto et al .
( 2005 ) in two chromacris species . c. nuptialis had only one bivalent ( m6 ) with a pericentromeric cma3 block , whereas c. speciosa presented cma3 blocks in two autosomal bivalents , a proximal one in m6 and a telomeric one in l2 . 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 .
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 ( vitturi et al . , 2002 ) , mollusks ( colomba et al . , 2002 ) ,
insects ( cabrero and camacho , 2008 ; cabral - de - mello et al . , 2011a ; panzera et al . , 2012 ) ,
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 .
we analyzed ten individuals of r. mariajoseae from gravat ( 081204 s ; 353353 w ) and bezerros ( 081400 s ; 354749 w ) and ten adult males of radacridium nordestinum from surubim ( 074959 s ; 354517 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 slides were stained with cma3 ( 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 g / ml ) for 20 min and mounted in glycerol / mcilvaine buffer / mgcl2 .
probes were obtained by pcr performed according to ayres ( 2002 ) , with modifications , using genomic dna from both species and the primers : 18s dnar - sca18s1f ( f 5 - ccc cgt aat cgg aat gag ta - 3 ) ; sca18s1r ( r 5- gag gtt tcc cgt gtt gag tc -3 ) , 5srdna f(5- aac gac cat acc acg ctg aa -3 ) ; r ( 5- aag cgg
tcc ccc atc taa gt - 3 ) , histone h4 f-1 ( 5 - tsc gig aya aca tyc agg gia tca c - 3 ) and r-1 ( 5 - cky tti agi gcr tai acc acr tcc at - 3 ) .
the pcr products analyzed after electrophoresis in a 1% agarose gel had the expected sizes .
the 18s rdna and histone h4 probes were labeled with biotin-16-dutp and the 5s rdna probe , with digoxigenin-11-dutp .
the chromosome preparations were submitted to an alcohol series pretreatment and then treated with rnase and pepsin .
the hybridization mix contained 1 l 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 .
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 ( l1-l3 ) , five medium ( m4-m8 ) and three small ( s9-s11 ) pairs .
cma3/da / dapi sequential staining showed gc - rich ( cma3 positive ) constitutive heterochromatin ( ch ) blocks in the interstitial region of only one medium - sized bivalent ( m5 ) and in the pericentromeric region of the x chromosome in r. mariajoseae ( figure 1a ) . in r. nordestinum ,
cma3 blocks were present in the interstitial region of the l2 bivalent and in the pericentromeric region of all chromosomes , except the l1 bivalent ( figure 1c ) .
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 l2 , s9 and s10 of r. nordestinum ( figure 2b ) .
the 5s rdna genes were located in the pericentromeric region of the two largest bivalents ( l1 and l2 ) and in the x chromosome of r. mariajoseae , ( figure 2c ) and in the bivalents l2 , l3 m4 and m5 of r. nordestinumm ( figure 2d ) .
the histone h4 probe was mapped in a proximal location in a medium - sized autosome bivalent ( m5 ) in both species ( figure 2e and f ) .
the karyotypic similarities detected after conventional analysis between the two species of radacridium did not extend to their cma3 and dapi staining patterns .
our results showed gc - richness ch heterogeneity . in r. mariajoseae only one autosome pair and the x chromosome presented cma3 blocks , which resembled the scarcity in gc - rich regions of other romaleidae , as two species of chromacris with a single cma3 block ( loreto et al . , 2005 ) .
the presence of a large number of cma3 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
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 ( agno3 ) by rocha et al . ( 1997 ) .
although three autosome bivalents showed 18s hybridization signals in r. nordestinum , only the l2 pair had a corresponding active nor identified by rocha et al .
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 .
, the ancestral rdna site could have been present in the m9 bivalent 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 m9 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 .
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 .
18s rdna sites have been frequently observed associated with gc - rich regions in romaleidae ( pereira and souza , 2000 ; loreto et al . , 2005 ) ,
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 ( m5 ) in both species .
a single chromosome pair bearing histone genes was also described in other species of grasshoppers .
( 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 l2 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 .
 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
Material and Methods
Results
Discussion