Different techniques involving fluorochrome staining, C-banding, NOR location and FISH were used in order to characterize the karyotype and to determine the characteristics of the constitutive heterochromatin in the genome of grasshopper Xestotrachelus robustus. This species presents uniform karyotype in terms of chromosome number (2n=23, XO in males) but deffered in the morphology of some chromosomes of the complement. Fluorescence in situ hybridization (FISH) was applied to the location of 45S genes. The results of FISH are compared with those coming from classical cytogenetics (C, AgNO3 and CMA3) banding procedures

HAVER, P. R. de O.

MELO, N. F. de

SOUZA, M. J. de

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CARYOLOGIA VoI.56, no. 3: 261-267, 2003Karyotype, C~and fluorescence-banding patterns,NOR Iocation and FISH in the grasshopperXestotracbelus robustas (Romaleidae)MARrAJOSÉDE SOUZA1"*,PATRíCIARoDRIGUESDEO;HAVERl-and NATONIELFRANKLINDEMEL02I Departamento-de-Genética, CCB, UnivelSidade Federal-de Pemambuco, Av. Prof. Moraes Rego, SN, Cidade Universitária,~Pem8mbuco, CEP 50732-970,..BrasiL2 Embrapa Semi-ârído, Petrolína, Brasil.Abstract- - Different· techniques involcing fluorochrome staining, C-banding,NORlocationandEISHwere.usediri.order to characterize the karyotype and todetermine the.characteristics of.the,constitutive heterochromatin in the genomeof grasshopperXesfotrachelus robustas. This species presents unifonn karyotypein tenns of chromosomenumber (2n=23,XOin males) but diffe.redin the rnor-phology of some- chromosomes of the complemento Fluorescence in situhybridization (FISH) was applied to the location of 45S genes. The results ofFISH are compared with thosecoming from classical cytogenetics (C, AgNO)and CMA)}banding procedures.Key words; constitutíve heterochromatin, NOR, C-banding, fluorochromes,FISH.INTRODUCTION'Xestotracbelas is an exelusively Neotropicalgrasshopper; This genus is taxonomically relatedto Chromacris. thatis polytypic and has a wide dis-tribution from Mexico to Argentina (ROBEKI'Sand CARBONEll1982). The Xestotracbelus phal-lic structures are similar to those in Cbromaais.One of the most frequent categories of chro-mosome differences.in grasshoppers involveschanges in the amount and localization of con-stitutive heterochromatin (JOHNand KING 1983;JOHN et al. 1985). C-banding offers informationconcerning karyotype structure and reveals dif-fere.nces inthe distribution of heterochromatin. Ithas been argued that heterochromatin is the mostdynamic of chromosome components and thisgenomic material is subject to both qualitativeand quantitative variation. Furthermore, the* Corresponding author: e-mail: mjslopes@Uol.com.brassortmenr ofheterochromatin and euchromatininto different chromosomal domains appears toplayalso an important role of chromosomal struc-ture (WALLRATII1998). FluorescentDNA-band-ing dyes of different specificity have made it pos-sible to characterize heterochromatin regions in amuch more precise manner. These fluorochromeshave been used whether they display AT-speci-ficity (DAPI) or GC-specificity (CMA)(SCH.WEIZER1976, 1981). Fluorochrome bright-ness is nonnally a function of DNA composition,modified by interaction with the chromosomestructure (SCHWEIZER1981).Nuc1eolus organizer regions (NORs) areimportant markers for the study of chromosomeevolution. The number and location of NORsare usually characteristic of species or popula-tion within different insect species (RUFASet ai.1987; BEDO 1991; BELLAet ai. 1993). In thisorganisms, the rRNA genes are clustered in oneor several nucleolus organizer regions (NORs)and usually located in heterochromatin.Karyolype, C-and ...2003 SP-PP-01289111111111111111111111111111111I1111111111111111111111111111111111I! IIIIIII!CPATSA-27353-1_. '-'-.J,J - 1.262 SOUZA, HAVER and MELOThe cytogenetics of the Neotropical acridoidgrasshoppers of the fami1yRomaleidae has beenstudied by some authors (VILARDI 1986, 1988;SoUZA and Sn.VA Fl:LHA 1993; SOUZA et aI. 1998;PEREIRA and SOUZA 2000). In most species hete-rochromatie C-positive bands were loealized inthe perieentromerie regions of the chromosomesalthough examples of non-eentromerie loeationwere also detected in some species (SOUZA andKmo 1995; ROCHA et aI. 1997). This paperdeserihes the charaeteristics of constitutive hete-crochromatin regions in meiotie and mitotie chro-mosomes of Xestotrachelus robustus using C-banding, specific fluoroehrome banding, NORloeation, as well as FISH of rRNA genes.MATERIAL AND METHODSA total of thirty individuais (21 males and 09females) of Xestotrachelus robustus were collected inthe states of Pemambuco and Bahia in the NortheastRegion of Brazil. Chromosome preparations wereFig. 1 -:-Standard karyotype and the pericentric inversion in the Me chromosome of X robustus. Mitoticmetaphase from ovariole cell and spermatogonial metaphase (a,b), respectively, (c) diplotene, (d) silver-staineddiplotene from males. AlI bivalents and X univalent show silver-stained kinetochores. Arrow indicares NORr.MElOSIS IN TIfE GllASSHOPPER XESTOTRACHEWS ROWS1US 263made fram testes of adult 'males fixed in 3:1ethanol:acetic acid. Females were treated withcolchicine (0.1 % in insect saline solution) in me pro-portion to 0.1 rnl/3g body weight, Cytological prepa-rations were made by the classical follicle squasbingmethod and chrornosomes were stained with 1% lac-toacetic orcein.. The Cbanding method was carriedout as described by SUMNER(1972). Silver stainingwas performed according to RUFAS et al. (19&5}..F1u-orescence patterns (CMA/DAJDAPI) were analyzedusing the teduúques developed by ScHwElzER: et ai.(1983) with minoromodifications. For sequential stain-.ing (AgNOiCMA,).the slides were stained with.silvernitrate, photogrephed, destained, stained withCMA3•~ .••.•• iawashed and mountedin a medium supplemented withglycerol. A probe containing fragments of ribosomalgenes 45S (18S-5.8S-25S) from Arabidopsis tbaliana(UNFRIEDet ai. 1989; UNFRIEDand GRUENDLER1990)was used. In situ hybridization procedure was fol-lowed according to MOSCONE et ai. (1996). Theprobes was labelled by nick translation (Life Tech-nologies) using biotin-ll-dU1P and detected usinganti-biotin-rhodamine. The slides were counterstainedwith DAPI and mounted in Vectashield (Vector Lab-oratories). Photographs were taken in a Leitz Ortho-plan microscope using Kodak T-MAX 400 and 400ISO Fuji Films. Copies were taken using Kodak.Kodabrome PríntF, paper.Fig. 2 - C-banding and fluorochrome staining in X. robustus. (a) C-banding plus Giemsa in diplotene.(b} C-bandingstained with acridine orange on diplotene. Triple staining CMA/DNDAPI in spermatogo-nial metaphaseIc.d), Note in (c) the pattem of CMA) positive in the entire chromosome complemento264 SOUZA, HAVER and MEtORESULTSThe conventional chromosome complementof Kestotracbelus robustus consists of2n=2J, XOin the males and 2n=24, xx: in the females. Theautosomes of this species were divided into threelarge (LIL}) andfive medium (~-M8) acrocen-tric pairs, two small meta-submetacentric pairs(S9-SIO) and one acrocentric pair (S11). TheXchromosomeís the4th in decreasing size (Fig.Ic),Three out six females and two males from Cha-pada Diamatina state of Bahia were heterozygousto a probably pericentric inversion in the Ma pairoOne male and two females from Buique state ofPemambuco were homozygous for this inver-sion. Thus, while one of the both Ms chromo-some appeared acrocentric, the other was sub-metacentric (Fig. la,b). The C-banding showedthat the chromosomes of X. robustas have hete-rochromatin restricted to pericentromeric regionsof all chromosomes of the complement (Fig.2a).111addítíon.the pairs S9 and SlO were almost com-pletely heterochromatic. The pattem of fluores-cent bands using acridine orange was the same ofthat found with Giemsa C-bands (Fig. 2b).The triple staining CMAiDA/DAPI revealednumerous bright CMA} blocks distributedthroughout the pericentromeric regions of allFig.3 - The sequential staining AgNO/CMA, on diplotene (a.b) respective-ly.Note the occurenceof GC-rich at the NOR (arrow), In (c) fluorescence in situhybridization of meiotk nuclei of X. robustus with the rDNA 455 probe. Theprobe hybridíze.wirh the pericentromerlc region of the.bivalent M, (arrow).MElOSIS IN TIIE. GRASSHOI.'I.'ERXESTOTRAaíEUI.I: ROBUSlUS.chromeseeees (Fig, 2e,d). X. robustus- showed-acomplete- eoiaeideaee of C-band pettems and·CMAJ+bends in-the-entire chromosome set, Nobright DNDAPI fluorescence was evident in thechromosome complement (Fig. zd), In mitoticmetaphase of females all chromosomes presentedthe maximal band resolution. One nudeolus wasobserved throughout the first meiotic prophase(Fig. 1d). The nudeolar remnants appeared asso-ciated to pericentromeric region of the M~ biva-lentoEarly diplotene cell in figure Id showed sil-ver-positive kinetochores in all bivalents and inthe single X chromosome. The FISH analysiswith the probe 45S rDNA revealed the intense ofrDNA sequences in the pericentromeric region ofbivalent M~. This label (Fig. 3c) ·corresponds tothe active NOR, located in this bivalent. Thesequenrial staining AgNO/CMA3 showed (Fig,Ja,b} that the heterochromatín associated withactive NORs appears bright with CMA} in thisspecies.DISCUSSIONRomaleidae is a very díversified Neotropicalfamily,with many species inhabiting arid or sub-arid areas. Most cytological data on this familywas obtained through conventional analysis(MEsAet alo1982)_The karyotype of K robustuscomprises nine pairs of acrocentric chromosomes(LI-~. ~-Ma and SII) and two meta-submeta-centric pairs (S9-SIO)plus a single X chromosomeín males and two Xin females. The.karyotypes ofromaleid species are remarkably uniform withregard to the number and morphology of theirchromosomes.. Furthermore, these apparent sim-ilarities in conventionally techniques disappearas more advanced methods of analysis areemployed. In X. robustus alI heterochromaticregions show DA/DAPI homogeneous stainingand DNCMA3 bright fluorescence. These cyto-Iogical data showed that the centromeric hete-rochromatin of the chromosomes is GC-rich. Inthis and other species, fluorescence staining tech-niques, provide an important method for map-ping heterochromatin of specific DNA composi-tion. This approach has been also demonstratedin several other species of grasshoppers (Gos-ALVESet aI. 1987; BELLAet aI. 1993). It permitsboth the characterization of heterochromatin andthe study of changes during the chromosomeevolution in this group of organisms. C-bandingpettems have been observed in few species ofthe-family Romaleidae. However, they haverevealed that significant intra and inter-specificdifferences in the distribution and amount ofconstitutive heterochromatin (VILARDI1986,1988; SOUZAand SILVAFILHA1993; SOUZAandKmo 1995; SOUZAet ai. 1998; PEREIRAandSoUZA2000). Furthermore, Xestotrachelus andCbromacris are two genera closely related. Cbro-macris speciosa, on the other hand, have largepericentromeric and telomeric blocks of consti-tutive heterochromatin (SOUZAand KIDo 1995),by comparison with X robustus that has basical-ly small pericentromeric blocks in all chromo-somes in the complemento In most extensivelycharacterized groups of acridid grasshoppersthere are examples where closely related specieswith similar karyotype differ in the heterochro-matin pattems (JOHNand KING 1983;JOHN etal.1985). Variation in the C-banding patterns amongcongeneric grasshopper are also reported by SAN-TOS et al. (1983) for Calliptamus, Oeidopoda andEuchorthippus and by ROCHAet al. (1997) forRadacridium.In X. robustus the 45S rRNA genes are clus-tered on single chromosomal site, located in thepericentromeric regions on bivalent M~. TheNORs on this bivalent were found to beAgN03/CMA} positive and marked to C-band-ing. This indicates that the pericentromericregions of this chromosome pair are mainly com-posed of constitutive heterochromatin. In con-trast, a different pattem of localization of therRNA genes has been found in the other roma-leid species (Xyleus angulatus) analysed (SOUZAetal. 1998). With respect to pericentric inversion inMa of X. robustus this chromosomal rearrange-ment occurs as a polymorphism. Evidences itdoes not lead in reproductive isolation and themeiotic pairing process is not disturbed. Thesmall number of specimens, and the limitedamount of testicular material examined does notallow to determine if the presence of the het-erozygous inversion had any effect on crossing-over frequency and locarion. On the other hand,small chromosomes (~-SlO) have also apparentlyundergone pericentric inversions without reduc-tion in the usual chromosome number (2n=23,XO in males). However, the probable pericentricinversions in this small chromosome have led toa modification of the chromosome morphology.These rearrangements could play a role in thespeciation processes of X. robustus. Likewise,266 SOUZA, HAVER and MELOpericentric inversion polymorplúsms have beendescribed in Australian morabines (WIDTE 1973;HEwrrr 1979). It is known that this kind of inver-sion also exists as fixed differences betweenspecies and may be involved in speciation (KING1993). Our results indicate that studies of a greatnumber of populations of X. robustus coveringthe whole range of this species are needed inorder to understand the role of the polymor-phism for pericentric inversion observed.Acknowledgements - We wish to thank DrMarcelo Guerra for laboratory facilities for FISHexperiment and also for critica1 reading of the man-uscript. We are gratefu1 to Dr. Carlos S. Carbonellfor the taxonomic identifieation of the speeies usedin this paper. This work has been suported by grantfrom Fundação de Amparo à Ciêneia e Teenologiado Estado de Pemambuco (FACEPE) and Consel-ho Nacional de Desenvolvimento Científico e Tec-nológico (CNPq).REFERENCESBEIJOD.G., 1991 - Cytological cbaracterization ofheterochromatin in mitotic and meiotic chromo-somes of tbe Old World screwworm fly,Chrysomya bezziana (Diptera: Callipbaridae).Genome, 34: 631-637.BELLAJ.L., SERRANOL., HEwrrr G.M. and GoS-ALVFZJ. 1993 - Heterocbromatin heterogeneityand rapid diuergence of the sex cbromosomes inCbortbippus parallelus parallelus and C. p. ery-tbropus (Ortboptera) Genome, 36: 542-547.GosALVFZJ., BELLAJ.L., LoPEl-FERNANDElC. andMEZZANOTIER, 1987 - Correlation betweenconstitutiue beterocbromatin and restrictionenzyme resistant cbromatin in Arcyptera tornosi(Ortboptera) Heredity, 59: 173-180.HEWITI G.M., 1979 - Ortboptera. Grassboppersand Crickets. In: B. John (Ed.), "Animal Cyto-genetie 3. Inseet". 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Karyotype, C-and fluorescence banding patterns, NOR location and FISH in the grasshopper Xestotrachelus robustus (Romaleidae).

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Karyotype, C-and fluorescence banding patterns, NOR location and FISH in the grasshopper Xestotrachelus robustus (Romaleidae).

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