INTRODUCTION

Xestotrachelus is an exclusively Neotropicalgrasshopper. This genus is taxonomically relatedto Chromacris that is polytypic and has a wide dis-tribution from Mexico to Argentina (ROBERTS

and CARBONELL 1982). The Xestotrachelus phal-lic structures are similar to those in Chromacris.

One of the most frequent categories of chro-mosome differences in grasshoppers involveschanges in the amount and localization of con-stitutive heterochromatin (JOHN and KING 1983;JOHN et al. 1985). C-banding offers informationconcerning karyotype structure and reveals dif-ferences in the 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

assortment of heterochromatin and euchromatininto different chromosomal domains appears toplay also an important role of chromosomal struc-ture (WALLRATH 1998). Fluorescent DNA-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 (CMA3)(SCHWEIZER 1976, 1981). Fluorochrome bright-ness is normally a function of DNA composition,modified by interaction with the chromosomestructure (SCHWEIZER 1981).

Nucleolus 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 (RUFAS et al.1987; BEDO 1991; BELLA et al. 1993). In thisorganisms, the rRNA genes are clustered in oneor several nucleolus organizer regions (NORs)and usually located in heterochromatin.

CARYOLOGIA Vol. 56, no. 3: 261-267, 2003

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

MARIA JOSÉ DE SOUZA1, *, PATRÍCIA RODRIGUES DE O. HAVER1 and NATONIEL FRANKLIN DE MELO2

1 Departamento de Genética, CCB, Universidade Federal de Pernambuco, Av. Prof. Moraes Rego, SN, Cidade Univer-sitária, Recife, Pernambuco, CEP 50732-970, Brasil.

2 Embrapa Semi-árido, Petrolina, Brasil.

Abstract - Different techniques involving fluorochrome staining, C-banding,NOR location and FISH were used in order to characterize the karyotype and todetermine the characteristics of the constitutive heterochromatin in the genomeof grasshopper Xestotrachelus robustus. This species presents uniform karyotypein terms of chromosome number (2n=23,XO in males) but differed in the mor-phology of some chromosomes of the complement. Fluorescence in situhybridization (FISH) was applied to the location of 45S genes. The results ofFISH are compared with those coming from classical cytogenetics (C, AgNO3and CMA3) banding procedures.

Key words: constitutive heterochromatin; NOR; C-banding; fluorochromes;FISH.

* Corresponding author: e-mail: mjslopes@uol.com.br

The cytogenetics of the Neotropical acridoidgrasshoppers of the family Romaleidae has beenstudied by some authors (VILARDI 1986, 1988;SOUZA and SILVA FILHA 1993; SOUZA et al. 1998;PEREIRA and SOUZA 2000). In most species hete-rochromatic C-positive bands were localized inthe pericentromeric regions of the chromosomesalthough examples of non-centromeric locationwere also detected in some species (SOUZA andKIDO 1995; ROCHA et al. 1997). This paperdescribes the characteristics of constitutive hete-

rochromatin regions in meiotic and mitotic chro-mosomes of Xestotrachelus robustus using C-banding, specific fluorochrome banding, NORlocation, as well as FISH of rRNA genes.

MATERIAL AND METHODS

A total of thirty individuals (21 males and 9females) of Xestotrachelus robustus were collected inthe states of Pernambuco and Bahia in the NortheastRegion of Brazil. Chromosome preparations were

262 SOUZA, HAVER and MELO

Fig. 1 – Standard karyotype and the pericentric inversion in the M8 chromosome of X. robustus. Mitoticmetaphase from ovariole cell and spermatogonial metaphase (a,b), respectively, (c) diplotene, (d) silver-staineddiplotene from males. All bivalents and X univalent show silver-stained kinetochores. Arrow indicates NOR.

made from testes of adult males fixed in 3:1 ethanol:acetic acid. Females were treated with colchicine(0.1% in insect saline solution) in the proportion to0.1 ml/3g body weight. Cytological preparations weremade by the classical follicle squashing method andchromosomes were stained with 1% lactoaceticorcein. The C-banding method was carried out asdescribed by SUMNER (1972). Silver staining was per-formed according to RUFAS et al. (1985). Fluores-cence patterns (CMA3/DA/DAPI) were analyzedusing the techniques developed by SCHWEIZER et al.(1983) with minor modifications. For sequential stain-ing (AgNO3/CMA3) the slides were stained with silvernitrate, photographed, destained, stained with CMA3,

washed and mounted in a medium supplemented withglycerol. A probe containing fragments of ribosomalgenes 45S (18S-5.8S-25S) from Arabidopsis thaliana(UNFRIED et al. 1989; UNFRIED and GRUENDLER 1990)was used. In situ hybridization procedure was fol-lowed according to MOSCONE et al. (1996). Theprobes was labelled by nick translation (Life Tech-nologies) using biotin-11-dUTP 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 KodakKodabrome Print F3 paper.

MEIOSIS IN THE GRASSHOPPER XESTOTRACHELUS ROBUSTUS 263

Fig. 2 – C-banding and fluorochrome staining in X. robustus. (a) C-banding plus Giemsa in diplotene.(b) C-banding stained with acridine orange on diplotene. Triple staining CMA3/DA/DAPI in spermatogo-nial metaphase (c,d). Note in (c) the pattern of CMA3 positive in the entire chromosome complement.

RESULTS

The conventional chromosome complementof Xestotrachelus robustus consists of 2n=23, XOin the males and 2n=24, XX in the females. Theautosomes of this species were divided into threelarge (L1-L3) and five medium (M4-M8) acrocen-tric pairs, two small meta-submetacentric pairs(S9-S10) and one acrocentric pair (S11). The Xchromosome is the 4th in decreasing size (Fig. 1c).Three out six females and two males from Cha-pada Diamatina state of Bahia were heterozygousto a probably pericentric inversion in the M8 pair.One male and two females from Buique state of

Pernambuco were homozygous for this inver-sion. Thus, while one of the both M8 chromo-some appeared acrocentric, the other was sub-metacentric (Fig. 1a,b). The C-banding showedthat the chromosomes of X. robustus have hete-rochromatin restricted to pericentromeric regionsof all chromosomes of the complement (Fig.2a).In addition, the pairs S9 and S10 were almost com-pletely heterochromatic. The pattern of fluores-cent bands using acridine orange was the same ofthat found with Giemsa C-bands (Fig. 2b).

The triple staining CMA3/DA/DAPI revealednumerous bright CMA3 blocks distributedthroughout the pericentromeric regions of all

264 SOUZA, HAVER and MELO

Fig. 3 – The sequential staining AgNO3/CMA3 on diplotene (a,b) respectively.Note the occurence of GC-rich at the NOR (arrow). In (c) fluorescence in situhybridization of meiotic nuclei of X. robustus with the rDNA 45S probe. Theprobe hybridize with the pericentromeric region of the bivalent M5 (arrow).

chromosomes (Fig. 2c,d). X. robustus showed acomplete coincidence of C-band patterns andCMA3+ bands in the entire chromosome set. Nobright DA/DAPI fluorescence was evident in thechromosome complement (Fig. 2d). In mitoticmetaphase of females all chromosomes presentedthe maximal band resolution. One nucleolus wasobserved throughout the first meiotic prophase(Fig. 1d). The nucleolar remnants appeared asso-ciated to pericentromeric region of the M5 biva-lent. Early diplotene cell in figure 1d 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 M5. This label (Fig. 3c) corresponds tothe active NOR, located in this bivalent. Thesequential staining AgNO3/CMA3 showed (Fig.3a,b) that the heterochromatin associated withactive NORs appears bright with CMA3 in thisspecies.

DISCUSSION

Romaleidae is a very diversified Neotropicalfamily, with many species inhabiting arid or sub-arid areas. Most cytological data on this familywas obtained through conventional analysis(MESA et al. 1982). The karyotype of X. robustuscomprises nine pairs of acrocentric chromosomes(L1-L3, M4-M8 and S11) and two meta-submeta-centric pairs (S9-S10) plus a single X chromosomein males and two X in 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 all heterochromaticregions show DA/DAPI homogeneous stainingand DA/CMA3 bright fluorescence. These cyto-logical 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-ALVES et al. 1987; BELLA et al. 1993). It permitsboth the characterization of heterochromatin andthe study of changes during the chromosomeevolution in this group of organisms. C-banding

patterns 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 (VILARDI 1986,1988; SOUZA and SILVA FILHA 1993; SOUZA andKIDO 1995; SOUZA et al. 1998; PEREIRA andSOUZA 2000). Furthermore, Xestotrachelus andChromacris are two genera closely related. Chro-macris speciosa, on the other hand, have largepericentromeric and telomeric blocks of consti-tutive heterochromatin (SOUZA and KIDO 1995),by comparison with X. robustus that has basical-ly small pericentromeric blocks in all chromo-somes in the complement. In most extensivelycharacterized groups of acridid grasshoppersthere are examples where closely related specieswith similar karyotype differ in the heterochro-matin patterns (JOHN and KING 1983; JOHN et al.1985). Variation in the C-banding patterns amongcongeneric grasshopper are also reported by SAN-TOS et al. (1983) for Calliptamus, Oeidopoda andEuchorthippus and by ROCHA et al. (1997) forRadacridium.

In X. robustus the 45S rRNA genes are clus-tered on single chromosomal site, located in thepericentromeric regions on bivalent M5. TheNORs on this bivalent were found to beAgNO3/CMA3 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 pattern of localization of therRNA genes has been found in the other roma-leid species (Xyleus angulatus) analysed (SOUZA etal. 1998). With respect to pericentric inversion inM8 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 location. On the other hand,small chromosomes (S9-S10) 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,

MEIOSIS IN THE GRASSHOPPER XESTOTRACHELUS ROBUSTUS 265

pericentric inversion polymorphisms have beendescribed in Australian morabines (WHITE 1973;HEWITT 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 critical reading of the man-uscript. We are grateful to Dr. Carlos S. Carbonellfor the taxonomic identification of the species usedin this paper. This work has been suported by grantfrom Fundação de Amparo à Ciência e Tecnologiado Estado de Pernambuco (FACEPE) and Consel-ho Nacional de Desenvolvimento Científico e Tec-nológico (CNPq).

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Received October 10, 2002; accepted January 28, 2003

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