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Genetica (2005) 125:253-260DOI 10.1007 Is 10709-005-0368-0

© Springer 2005

A comparative cytogenetic analysis between lhe grasshopper speciesChromacris nuptialis and C. speciosa (Romaleidae): constitutiveheterochromatin variability and rDNA sites

Vilma Loreto ':", Eva Stadtler ', Natoniel F. de Melo2 & Maria José de Souza'IDepartamento de Genética, CCB, Universidade Federal de Pernambuco (Phone: + 55-81-2126-8520; E-mail:vloreto@zipmail.com.br), 2Embrapa Semi-àrido, Petrolina, Brasil, tAv. Prol Moraes Rego sln CidadeUniversitária, Recife, PE, 50732-970, Brasil

Received 3 April 2005 Accepted 23 June 2005

Key words: FlSH, grasshopper, heterochromatin, karyotype, NORs

Abstract

The chromosomes of Chromacris nuptialis and C. speciosa were comparatively analyzed using differentcytogenetic techniques, in order to determine the level of karyotypic similarities and differences between thespecies. The results show similarities in chromosome number (2n = 23,XO) and acrocentric morphology.lnsome C. nuptialis individuais meiotic irregularities were detected involving the L2 bivalent. This bivalent wasdelayed and presented anaphasic bridges and other aberrations. Differences in constitutive heterochromatin(CH) patterns and composition were observed through C-banding and fluorochromes staining. Silver nitratestaining revealed a single medium nucleolar organizer regions (NORs) pair, per species. Differences were alsoobserved in NORs location, which was pericentromeric in C. nuptialis and proximal in C. speciosa. FISH usingan rDNA probe confirtned the existence of ribosornal sites coinciding with active regions visualized by silvernitrate. The possible implications of the karyotype differences observed between both species are discussed.

lntroduction

Grasshoppers of the genus Chromacris are re-stricted to the Neotropical region and are foundfrom Mexíco to Argentina. These insects presentstrong colors wíth yellow or red stains, on theirwings. Based on those characteristics, Robertsand Carbonell (1982) divided the genus Chroma-cris in two groups: one called trogon, that in-cludes C. trogon, C. psittacus, C. icterus andC. peruviana, and another called colorata thatconsists of C. colorata, C. minuta, C. miles, C.speciosa and C. nuptialis. The latter taxon is re-lated to C. speciosa, which was ínitially consideredto be a variant formo However, the study of somemorphological traits has indicated that it is adistinct species (Roberts & Carbonell, 1982).

Romaleídae famíly has shown a marked pre-dominance of conserved karyotypes (2n = 23,24

A comparalive cylogenelic ...2005 SP-PP-00238111111111111111111111111111111111111111111111111111111111111111111111111111

CPATSA-32721-1

XO:XX) (Mesa, Ferreira & Carbonell, 1982). Nev-ertheless, analysis of constitutive heterochromatin(CH) by C-banding has revealed both variations interms of the quantity and location of CH blocks,among different species. Pericentromeric locationhas been the most frequent having been observed inseven out of eight species studíed so far (Vilardi,1986; Souza & Kido, 1995; Pereira & Souza, 2000;Souza, Haver & Meio, 2003). Additionaly, ocur-rence of interstitial and distal blocks has been re-ported for Xyleus angulatus (Souza & Kido, 1995;Souza, Rufas & Orellana, 1998), Radacridiummariajoseae, R. nordestinum (Rocha, Souza & Tas-000, 1997), and Chromacris speciosa (Souza &Kido, 1995). Polymorphisms for supernumeraryheterochromatic segments have been described forX. angulatus (Souza & Silva-Filha, 1993) and R.nordestinum (Rocha, Souza & Tashiro, 1997) and Bchromosomes have been observed in X. angulatus

254

(Souza & Kido, 1995) and Zoniopoda tarsata (Vi-lardi, 1986), proving the intensive varia bility in CH,among representatives of this family.

As to cytogenetic characteristics, knowledgeabout the genus Chromacris is limited to thestudies of Mesa, Ferreira and Carbonell (1982)who described the karyotype of three species (C.speciosa, C. peruviana and C. milesy as 2n = 23,XOand 2n = 24,XX and Souza and Kido (1995) thatanalyzed the variability in CH distribution in C.speciosa by C-banding.

Some studies have shown that comparativecytogenetic analysis of grasshoppers can contrib-ute to the understanding of the speciation process(Bella et aI., 1990). Bridle et al. (2002) emphasizedthe close karyotype similarity between Chorthip-pus brunneus and C. jacobsi, when analyzingC-banding patterns and CH composition by fluo-rochrome labeling. However, Rodriguez-Ifíigo,Bella and García de Ia Vega (1993), analyzingtwo species of the genus Dociostaurus, and Cam-acho and Cabrero (1983), analyzing the speciesAcrotylus insubricus and A. patruelis, showed dif-ferences in the patterns and composition of CHbetween related species, indicating that bothloss or acquisition of heterochromatin might beinvolved in karyotype evolution.

ln the present study, we performed a com par-ative cytogenetic analysis between C. nuptialis andC. speciosa by conventional analysis, C-banding,fIuorochrome and silver nitrate staining, and flu-orescence in situ hybridization (FlSH) using a 45SrDNA probe, in order to determine the leveI ofsimilarities and differences between these species.

Material and methods

A total of 16 adult male specimens of C. nuptialiscollected in the locality of Exu (7°26'0" S39°50'36" W) and 25 specimens of C. speciosacollected in Moreno (8°7'7" S 35°5'32" W) (15males and 3 females) and Igarassú (7°50'3" S34°54'23" W) (5 males and 2 females), State ofPernambuco, Brazil, were studied. The grasshop-pers were brought to the laboratory, dissected andthe testes and ovaries were fixed in ethanol:aceticacid (3:1). Females were injected with 0.1 % col-chicine for 6 h. Slides were prepared using theclassical testicular and ovarian follicles squashingtechnique, followed by staining with 2% lacto-

acetic orcein for conventional chromosome anal-ysis. For the different banding techniques, theslides were prepared by the addition of one drop of45% acetic acid and were covered with coverslipsthat were removed after freezing in liquid nitrogen.

C-banding and CMA3jDAjDAPI staining

C-banding was performed according to Sumner(J 972). Slides aged for 2 days were treated with0.2 N HCI for 30 min, 5% barium hydroxide (60°C)for 45 s, and 2x SSC (60°C) for 45 mino After dry-ing, the preparations were stained with Giemsa 5%diluted in phosphate buffer, pH 7.0, for 3 mino ForCMA3jDAjDAPl staining (Schweizer et al., 1983),the slides were aged for 3 days, stained with CMA3

(0.5 mgjml in McIlvaine buffer, pH 7.0) for I h,washed in distilled water, stained with DistamycinA (DA) (0.1 mgjml) for 45 min, washed again, andstained with DAPl (0.5 /lgjml) for 20 mino

Silver nitrate staining (AgN03) and fluorescence insitu hybridization (FISH)

Cytological preparations were pretreated with 2xSSC at 60°C for 10 min, washed, dried, stainedwith one drop of AgN03 solution (1:1) in distilledwater (with the pH adjusted with formic acid), andthen incubated in a humid chamber at 70°C for3 min (Rufas, Esponda & Gosálvez, 1985). Somepreparations were counterstained with Giemsabefore mounting.

For FISH, a probe containing fragments ofthe45S ribosomal genes (18S-5.8S-25S) of Arabidop-sis thaliana (Unfried, Stocker & Gruendler, 1989;Unfried & Gruendler, 1990) was used to localizerDNA sequences. The probe was labeled withbiotin=I l-d UTP by nick translation, and thepreparations were incubated with RNAse, pro-teinase K, MgCI2jPBS and paraformaldehydejPBSsolutions, and final1y dehydrated in an increasingalcohol series (70-90-100%, 5 min each) accord-ing to Moscone, Matzke and Matzke (1996).Hybridization was performed at 80°C using 5 ul ofthe mixture. The slides were kept at 37°C over-night for renaturation. The probe was detectedwith an anti-biotin-rhodarnine antibody. Thechromosomes were counterstained with DAPI andthe slides were mounted in Antifade Vectashield(Vector) medium.

Photographs were taken with a Leica miero-scope using Kodak ISO 25 films, TMAX -400 andFuji Film lSO-400 for FlSH, and printed onKodak Kodabrome Print F3 paper.

Results

The diploid number of Chromacris speciosa andC. nuptialis was 2n = 23,XO, in males and2n = 24,XX, in females. Both species presentaeroceutrie ehromosomes whieh were grouped intwo large sized oue (L1-L2), six medium (MrMs)and three small pairs (S9-SI d. The X chromosomewas medi um sized (Figure la-t).ln both species, theX presented a variable heteropycnotic behavior,

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255

positive from leptotene to diplotene and negativeduring diakinesis and metaphase 1(Figure la-c).

Our cytogenetics studies in the 16 individuaisof C. nuptialis confirmed that six of them havemeiotic irregularities likely the stickiness. TheL2 pair presented anaphasic delay and atypicbridges formation through the union of thehomologous chromatids, in terminal position(Figure 2a-b). ln that region a block of con-stitutive heterochromatin (CH) was observedclose to the sticky point (Figure 2b). ln somecelIs the behavior of the laggard pair may indi-cate that it has not been ineorporated to the newformed nucleus (Figure 2e). The presence ofehromosomal fragments (Figure 2d) might haveoriginated from the separation of telophasic cells

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Figure l . Meiotic cells of Chromacris nuptialis (a.b.d.f) and C. speciosa (c and e). (a) Pachytene, (b) Diplotene, (c) Metaphase I, (d)Anaphase I, (e) Metaphase 11 with 11 chromosomes, and (I) Anaphase II with 12 chromosomes. Note the X chromosome hetero-pycnotic positive (a and b) and negative (c). Bar = 10 fim.

256

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Figure 2. Irregularities in the meiosis I of C. nuptialis. (a and b) Anaphase showíng the bridge formation among homologous chro-matíds of bivalent ~. Note in (b) the terminal block of CH in ~ (arrow). (c and d) Telophase showing the delayed pair and lheunion among chromatids resulting in fragments. Bar = 10 J.Im.

and the breaking of bridges. Out of the 647 celisin anaphase and telophase 1 from three individ-uaIs analyzed, 15 (2.3%) presented bridge for-mation and 632 (97.7%) were normal.

C-banding allowed the visualization of distinctCH distribution patterns in the two species.C. nuptialis exhibited CH blocks in the pericen-tromeric region of most chromosomes, while thesmali pairs presented variable CH blocks in loca-tion and size. lt was visualized an interstitial, atelomeric and a small block and apparently peri-centromeric in the S9, SIO and SI' bivalents,respectively. A block was also observed in thetelomeric region of the L2 pair (Figure 3a).

On the other hand, C. speciosa, presented ahighly variable CH, in terms of size and blockposition. C-banding of mitotic metaphasesrevealed pericentromeric CH in ali chromosomes.However, on M3, M4, M, and M6 pairs CH blockscovered a marked portion of the proximal part ofthe long armo A telomeric C-band was observed onthe L, and L2 pairs, and a large interstitial blockwas seen in the Ms pairo A small intersticial block

was also noted on the M, and M7 pairs, as well asin the X chromosome (Figure 3c). This patternwas detected in meiotic cells (Figure 3b).

The CMA3/DA/DAPI staining revealed differ-ent patterns in the species. Whereas C. nuptialispresented only one bivalent (M6) with a CMA;-block in the pericentromeric region (Figure 3d), C.speciosa had two CMA;- blocks, one in the proxi-mal portion of M6 and another in the telomeric re-gion of L2 (Figure 3f). DAPI staining washomogenous in the two species (Figure 3e,g).

Silver nitrate staining identified only oneNOR, in the medium pair (M6) of both species(Figure 4a, c). In situ hybridization with therONA probe confirmed the existence of only onepair of ribosomal sites per species (M6) and thesesites were located in different regions of thechromosomes, with a pericentromeric locationbeing observed for C. nuptialis (Figure 4b) and aproximal location for C. speciosa (Figure 4d). lnboth cases, the nucleolar organizer regions(NORs) coincided with the CMA;- heterochro-matin regions.

257

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Figure 3. C-banding pallern in C. nuptialis (a) and C. speciosa (b and e). Note the CH blocks on the srnaU pairs and in ~. CMAJ!DAfOAPI staining of diplotene cells from C. nuptialis (d and e) and C. speciosa (f and g). The arrows indicate lhe CMA; blocks,Bar = 10 um,

Discussion Souza & Tashiro, 1997). One exception to thisuniformity has been described for Xestotrachelusrobustus (2n = 23,XO and 2n = 24,XX), a speciestaxonomically related to Chromacris, where the ~and SIO pairs are meta-submetacentric and prob-ably originated from a pericentric inversion, sincethe rearrangement did not change the chromo-some number of the species (Souza, Haver &

Karyotypes of C. nuptialis and C. speciosa resem-ble those reported for other Chromacris speciesand for most romaleid species, as to chromo-some number (2n = 23,XO and 2n = 24,XX)and morphology (acrocentric) (Mesa, Ferreira &Carbonell, 1982; Souza & Kido, 1995; Rocha,

258

(a)

. ".

Figure 4. Identification of nucleolar organizer regions (NORs) and rDNA sites in C. nuptialis and C. speciosa by siJver nitrateslaining (a and c) and FISH (b and d), respectively. The arrows indicate lhe M,; pair with NORs in lhe pericenlromeric region ofC. nuptialis (a and b) and in lhe proxirnal region of C. speciosa (c and d). Bar = 10 pm,

Meio, 2003). lndeed, romaleid species have showna highly conserved karyotype, with only a fewcases of rearrangements being known (X-auto-some and autosome-autosome fusion), what leadto a reduction in chromosome number (Mesa,Ferreira & Carbonell, 1982).

Cellular events as paracentric inversion (Koeh-ler et aI., 2002), cohesion loss among chromatid-sisters (Cimini et al., 2003) and faiJure in thecheckpoint of the metaphasejanaphase control(LeMaire-Adkins, Radke & Hunt, 1997) has beendescribed in some organisms. However, the detailedanalysis of meiotic aberrations in C. nuptialis doesnot seem to indicate any of those events. A possibleexplanation for the abnormalities would be that thesticky among homologous chromatids to the blockof CH of L2 is maintained by proteins connection inthe heterochromatin. Stickness has been describedin polytene chromosomes of Drosophila andChironomus, in mitotic chromosomes of maizecallus culture (Fluminhan, Aguiar-Perecin & DosSantos, 1996) and in Ornithogalum longibracteatum(Pedrosa et al., 2001), where the existence ofproteicfactors, associated with CH, was admitted.

The karyotype similarity detected by conven-tional analysis between romaleid species was notobserved when comparing CH pattems. No simi-lar pattem has been identified between the few

species studied thus faro Despite a predominanceof pericentromeric CH, a wide variability in size,block locations (telomeric, interstitial and proxi-mal) and the presence of an extra CH as super-numerary segments and B chromosomes, havebeen reported (Vilardi, 1986; Souza & Kido, 1995;Rocha, Souza & Tashiro, 1997; Pereira & Souza,2000; Souza, Haver & Meio, 2003). When CHpattems of C. nuptialis and C. speciosa werecompared with those of Xestotrachelus robustus, acloser similarity was observed between C. nuptialisand X. robustus due to the predominance of peri-centromeric CH. However, differences were de-tected ín the small pairs. Whereas C. nuptialispossesses an interstitial block in S9, a telomeric inSto and a pericentromeric one in Silo in X. robustusthe S9 and Sto chromosomes are entirely hetero-chromatic (Souza, Haver & Meio, 2003). Chro-macris speciosa showed even a more particularpattem consisting of large pericentromeric CHblocks, on the large and medium bivalents, and adiversity of proximal, telomeric and interstitialblocks. This divergence of C-banding pattem inChromacris indicates that heterochromatín rear-rangements (amplification for example) may beinvolved in the karyotypic evolution of the group.

Comparison of the results obtained by CMA3/

DA/DAPI staining for C. nuptialis and C. speciosa

also revealed some differences. Chromacris nup-tialis had only one CMAt pericentromeric block,while two blocks (a proximal and a telomeric one)were observed ín C. speciosa. Thís scarcity of GCbase paírs ín CH contrasts wíth data reported forother romaleid species, in which CMAt blockspredominate (Souza, Rufas & Orellana, 1998;Pereira & Souza, 2000; Souza, Haver & Meio,2003). John et al. (1985), analyzing ten Australianacridid grasshoppers, identified distinct classes ofCH, when cytological preparations of these specíeswere staíned wítb CMA3 and DAPl f1uoro-chromes. ln Recitropis sp.l a part of CH wasCMAt, while the other was homogenous for thesef1uorochromes. On the other hand, Ursina sp.showed three classes, CMAt, DAPl + and a thirdone wítbout specíficíty. Two categories of CHcould be distinguíshed in the species analyzed here,one GC-rich and the other, a more frequent, onewithout any specific richness.

The location of NORs in representatives of theRomaleídae family can be dívided into three cat-egories: those restricted to autosomes, as observedin Xestotrachelus robustus (perícentromeric regionof Ms) (Souza, Haver & Meio, 2003), Radacridíumnordestinum (interstítial region of the L2 bívalent)(Rocha, Souza & Tashiro, 1997) and Phaeopariamegacephala (proximal region of M6) (Pereira &Souza, 2000); those located on the sex chromo-some, as in Radacridium mariajoseae (proxímal siteof the X) (Rocha, Souza & Tashiro, 1997); andthose wíth NORs on botb autosomes and allo-somes, as ín Xyleus angulatus, with proximal siteson the L3 and M4 bivalents and the X chromosome(Souza, Rufas & Orellana, 1998).

Chromacris nuptialis and C. speciosa had on1yone nucleolar organizer pair, as observed for mostromaleíd species analyzed, but they differed interms of the NOR position, which was pericentrícin C. nuptialis and proximal in C. speciosa. Sincethe NOR is located on the same pair (M6) in thosespecies, we may suppose that some event (para-centric inversion, heterochromatin amplificationor unequal crossing-over) might have Ied to themodification of the original pericentric position tothe proximal one, observed in C. speciosa duringthe course of its evolution. Paracentric inversionwas hyphothesized as the rearrangement involvedin karyotypes differences for NORs location intwo fish species. The location was in pericentro-meric and subtelomeric region, in Umbra pygmaea

259

and U. limi, respectively (Rab et al., 2002).Moreover, amplification was pointed out as thefirst event, in the different NORs location inTriticeae genomes (Dubcovsky & Dvorak, 1995).

FlSH using 45S rDNA probe confirmed theexistence of only one nucleolar organizer pair inthe two species and helped to clarify the positionof rDNA sites, which are sometimes hidden bynucleolar remnants, when visualized by silver ni-trate. This result is similar to the one reported bySouza, Haver and Meio (2003) for X. robustusshowing only one sign on M, pairo Since FlSHcoincided witb tbe proximal region of the M6bivalent (CMAt) of C. speciosa, and no sign wasobserved in the CMAt telomeric region of ~, tbeCH in this region, although GC-rich, is likely toshow no functional relationship with rDNA sites,and it might be organized differentially from theother CH regions.

Our results indicated a significant degree ofchromosome differentiation between C. nuptialisand C. speciosa, when comparing the distributionpattem and composition of CH, as well as theposition of the rDNA sites. The karyotype differ-ences observed indicate a distinction in taxonomicstatus between species, confirming the analysis ofsome morphological traits, performed by Robertsand Carbonell (1982), who demonstrated that C.nuptialis is not a variant form of C. speciosa.However, only cytogenetic and molecular analysisof other members of colorata and trogon groupswill determine whether the differences observed arefrequent among these representatives, or whetherC. speciosa and C. nuptialis can be íncluded indistinct groups.

Acknowledgements

We are very grateful to Dr Carlos S. Carbonell,Montevídeo University, for the taxonomic ídenti-fication of the species and Prof Dr MarceloGuerra for laboratory facílities for FISH experí-ment and for his opinion and valuable commentson the manuscript. The authors also grateful toDr Neide Santos, Dr Rita de Cássia de Mouraand Dr Aline Alexandrino for helpful suggestionsand the revision of the manuscript. To FranciscaTavares for technical support. This research wassupported by CAPES (Coordenação de Aper-feiçoamento de Pessoal de Nível Superior),

260CNPq (Conselho Nacional de DesenvolvimentoCientífico e Tecnológico) and FACEPE (Fun-dação de Amparo à Ciência do Estado de Per-nambuco). Eva Stadtler was student of scientificinitiation (PIBlqCNPq).

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