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Hereditas 138: 200-206 (2003)

Karyotypic characterization of representatives from Melolonthinae(Coleoptera: Scarabaeidae): karyotypic analysis, banding andfluorescent in situ hybridization (FISH)RITA DE CÁSSIA DE MOURAI.2, MARIA JOSÉ DE SOUZAi, NATONIEL FRANKLIN DE MEL03

and AMARO DE CASTRO LIRA-NETO I

I Departamento de Genética, Centro de Ciências Biológicas, Universidade Federal de Pernambuco, UFPE,Brasil2 Departamento de Biologia, Instituto de Ciências Biológicas, Universidade de Pernambuco, UPE, Brasil3 Empresa Brasileira de Pesquisa Agropecuária, EMBRAPA, semi-árido, Petro/ina, Brasil

Moura, R. c., Souza, M. J., Meio, N. F. and Lira-Neto, A. C. 2003. Karyotypic characterization of representatives fromMelolonthinae (Coleoptera: Scarabaeidae): karyotypic analysis, banding and fíuorescent in situ hybridization (FISH).-Hereditas 138: 200-206. Lund, Sweden. ISSN 0018·0661. Received April 25, 2002. Accepted June 30, 2003

Meiotic chromosomes of Phy//ophaga (Phytalus) vestita, Phyllophaga (Phy//ophaga) ali capillata and Lyogenys fuscus(Melolonthinae) were analyzed by conventional staining, Cbanding, ftuorochrornes, silver nitrate and FISH. The threespecies had a diploid number of 2n =20 and a sex mechanism of the (XyP; XY~ parachute type. P. (Phytalus) vestita,P. (Phy//ophaga) ali capillata and Lyogenys fuscus showed pericentromeric constitutive heterochromatin (CH) in aliautosomal bivalents and on X chromosomes. Staining with CMA3 and DAPI ftuorochrornes showed that lhe CH of P.(Phytallls) oestita is not specifically rich in AT and GC-base pairs, whereas in P. (Phy//ophaga) ali capillata the sexbivalent and one autosomal pair were found to be enriched in GC base pairs with CMA3, and in Lyogenys [uscus CH waspositive for DAP!. Silvernitrate staining revealed nucleolar remnants in al1 three species. However, FISH obtained aprecise identification of nucleolar organizing regions with an rDNA 18S and 25S probe. A signal of hybridization wasseen in each species, being detected in the X chromosome of P. (Phytalus) vestita and Lyogenys fuscus, and in a smallautosomal bivalent of P. (Phy//ophaga) ali capillata.

Rira de Cássia de Moura, Departamento de Genética, Centro de Ciências Biológicas, Universidade Federal de Pernambuco,UFPE, Av. Prof. Moraes do Rego S/N, Cidade Universitária, CEP: 50732-970, Recife, Pernambuco, Brasil. E-mail:mourarita@bol.com.br

The superfamily Scarabaeoidea is represented by 13families the world over, among them, the Scarabaei-dae family is particularly important by having under-gone wide adaptive spreading, with a faunacomprising approximately 2000 genera and 25000species. A total of 362 genera and 4706 species havebeen recorded in the Neotropical region, and about1777 species belonging to 204 genera have beenrecorded in Brasil (COSTA 2000).

Although Scarabaeidae represents a rich and diver-sified fauna, chromosomal studies of this family arestill scarce. About 323 species have been studiedkaryotypically thus far, corresponding to 1.29 % ofthe species described (SMITH and VIRKKI 1978; YA·DAV and PrLLAI 1979; YADAV et al. 1979; VIDAL andNOCERA 1984; COLOMBA et aI. 1996, 2000a; BIONE1999). A considerable predominance of a diploidnumber of 2n = 20, biarmed chromosomes and a sexmechanism of the XYp type has been widely detected.

The use of C-banding, silver nitrate staining andbase-specific fluorochromes is limited to fewscarabeoid species, such as Enema pan (VIDAL andGIACOMOZZI 1978), Glyphoderus sterquilinus, Eucra-nium arachnoids, Anomiopsoides heteroclyta (VIDAL

and No CERA 1984), G. sterquilinus, Bubas bison(COLOMBA et al. 1996), Macraspis festiva, Pelidnotapa//idipennis, Lygyrus ebenus, Geniates bore//i (BIONE1999), and Gymnopleurus sturmi (COLOMBA et al.2000a). On the other hand, fluorescent in situ hy-bridization (FISH) with rDNA probes has been usedin a few beetles species (GÁLIAN et al. 1995; DE LARÚA et al. 1996; PETITPIERRE 1996; COLOMBAet al.2000a).

The aim of this study is to investigate the meioticchromosomes of males of the species Phyllophaga(Phytalus) vestita, Phyllophaga (Phy//ophaga) affcapil/ata and Lyogenys fuscus using conventionalstaining, differential chromosome banding techniquesand fluorescent in situ hybridization (FISH). The useof these techniques permitted a compara tive analysisof the three Melolonthinae species studied.

MATERIAL ANO METHODS

The number and sources of the specimens studiedwere as follows. Phyllophaga (Phytalus) vestita (10specimens), Phy//ophaga (Phy//ophaga) aff capillata(12) and Lyogenys fuscus (15) were collected in difTer-

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Hereditas 138 (2003) Karyotypic characterization of Melolonthinae 201

ent areas of the Atlantic Forest in the State ofPernambuco, northeastern Brazil.

Male gonads from adult beetles were fixed mCarnoy (ethanol and acetic acid, 3:1) and used toobtain meiotic chromosomes. Cytological prepara-tions were obtained by the c1assicalsquashingmethod and the chromosomes were stained with 2 %lactoacetic orcein. C-banding was performed follow-ing SUMNER (1972), with some slides being stainedwith Giemsa (CBG) and other with DAPl (CBfDAP1). Triple staining with CMA3fDAfDAPl wasperformed according to SCHWEIZERet a!. (1983) anddouble staining with CM'A3fDA and DAPlfDA wasalso applied in the threespecies. Silver nitrate stain-ing (AgN03) was performed by the method of RUFASet aI. (1987). ln the fluorescent in situ hybridization(FISH) we used l8S and 25S probes (rDNA) ofArabidopsis tha/iana (UNFRIED et a!. 1989; UNFRIEDand GRUNDLER 1990) and thetechnique ofMOSCONE et aI. (1996). The probes were labeled withbio-ll-dUTP bynick .translation (Life Technologies)and detected by rat antibiotin antibodies (DakopattsM0743, DAKO) and anti-rat antibodies (DakopattsR0270, DAKO) produced with a rabbit TRITC (te-tramethyl-rhodamine isothiocyanate) conjugate. Thepreparations were counterstained with DAPI (2 ~gfml) and mounted with Vectashield H-IOO(} (Vector).

Slides were examined with a Leitz Orthoplan pho-tomicroscope. Conventional staining, fluorescenceand FISH were photographed with Kodak Imagelink

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a b

ASA 25 film, T-MAX 400 ASA film and Kodacolor400 ASA film (Kodak), respectively.

RESULTS

Conventional staining

Phyllophaga (Phytalus) oestita, Lyogenys fuscus andPhyllophaga (Phy/lophaga) aff capillata presented adíploíd number of 2n = 20, meioformula 9Il + XYP'ín the former ones, beíng 91I +XY p in the P. (Phyl-lophaga) aff cap illa ta , sex-determining mechanisms ofthe parachute type and an achiasmatic associationbetween the sex chromosomes. All the three specieshave symmetrical karyotypes and apredominance ofapparently biarmed chromosomes, as observed inmetaphases I (Fig. Ia-c). The male sex chromosomesare represented by a heteromorphic pai r, which forma typical parachute in P. (Phytalus) uestita (Fig. Ia)and L. fuscus (Fig. 1c), with a very small X chromo-some and a punctiform Y chromosome (Xy.). In P.(Phyllophaga) aff capillata the sex chromosomes(XY p) are small and present identical size (Fig. 1b).

C-banding and fluorochrome staining

The localization of constitutive heterochromatin(CH) blocks was defined by C-banding only in P.(Phytalus) vestira, which presented pericentrornericblocks in all autosomes. This pattern permitted theidentification of the occurrence of biarmed chromo-somes in this species. The sex bivalent was almost

Fig. la-c.Conventionalstaining in the three Melolonthinae species.Metaphases I. in Phyllophaga(Phyta/us) vestita (a), P. (Phyllophaga). ali capillata (b) and Lyogenys fuscus (c). Note the parachuteconfiguration of the sexual bivalent XyP and XYp (arrows). Bar = 10 JlII1.

202 R. de Cássia de Moura et ai. Hereditas138 (2003)

fully heterochromatic (Fig. 2a-b). In contrast, for P.(Phyllaphaga) aff capillata (Fig. 2e) slides pretreatedby C-banding and stained with DAPI (CB-DAPI)showed the presenee of apparently smaller CH blockscompared to P. (Phytalus) vestira. Triple stainingwith CMA3/DA/DAPI in P. (Phytalus) oestita didnot reveal any differential amount of CH and showedthat all blocks were labeled both by CMA3+ andDAPI + (Fig. 2c-d). However, DAPI labeling wasmore intense than CMA3. In P. (PhylIophaga) affcapillata double staining with CMA3/DA showed thepresence of CMA3+ blocks only in the sex bivalentand in a small autosomal bivalent (Fig. 2f). NoDAPI + blocks were detected in this species (resultnot shown). On the other hand, the CH of the entirechromosome complement in L. fuscus showed homo-geneous CMA3 staining (Fig. 2g) and strongly posi-tive DAPI staining (Fig. 2h), clearly showing that theCH is AT-rich.

AgN03 staining and fiuorescent in situ hybridization(FISH)

Silver nitrate staining showed the presence of nucle-olar remnants in only one chromosome pair of eachspecies. In P. (Phy//ophaga) af! capillata the activeNOR was observed in an autosome (Fig. 3c), incontrast to P. (Phytalus) vestita (Fig. 3a) and L.fuscus (Fig. 3e) in which the NORs were located inthe sex bivalents. The sex bivalent was stronglystained by silver and continued to be labe1ed duringthe different phases of meiosis, as can be seen in P.(Phyllophaga) aff capillata and L. fuscus (Fig. 3g-h).Silver nitrate also 1abe1edthe CH of the autosomalbiva1ents (Fig. 3g-h). In meiotic bivalents FISH withthe 18S and 25S ribosoma1 probe (rDNA) onlyshowed one signal of hybridization in each species(Fig. 3b,d,f), confirroing the AgN03 staining. Never-theless, in P. (Phy//ophaga) af! capillata (Fig. 3d),labeling oceurred in a small-sized autosome, whereasin P. (Phytalus) vestita (Fig. 3b) and L. fuscus (Fig.3f) the hybridization signals were present in the Xchromosome.

DISCUSSION

The karyotypic constitution of 2n = 20,Xyp in males(meioforroula 9II +Xy.) in Phyllophaga (Phytalus)vestita, Phy/lophaga (Phly/lophaga) aff capillata andLyogenys fuscus corresponds to the karyotype mostfrequently encountered in the fami1y Scarabaeidae.This karyotype has also been detected in more than50 % of the Melolonthinae species analyzed thus far(SMITHand VIRK.K.l1978; YADAVand PILLAI 19761979). Although this subfamily is considered to bechromosomally conserved, generally presenting

biarmed autosomes and a sex mechanism of theparachute type (Xyp), some chromosome rearrange-ments have led to changes in chromosome morphol-ogy, size and diploid numbers. Some examples areApogonia sp with 2n = 21,XO (SAHA1973), Apogonianigricans and Apogonia sp with 2n = 19,XO (MANNAand LAHIRI 1972; YADAV and PILLAI 1974) andOphthalmoserica karafutoensis with 2n = 18 (KUDOHet aI. 1973).

Although C-bands are localized in the pericen-tromeric region of most of the Scarabaeidae speciesanalyzed (BIONE 1999; COLOMBAet aI. 2000a), asalso observed here in P. (Phytalus) vestita and L.fuscus, other scarabeoids such as Enema pan (VIDALand GIACOMOZZI1978), Glynoderus sterquilinus andEucranium arachnoides (VIDAL and No CERA 1984)show interstitia1 and telomeric C-bands. This has a1sobeen reported for representatives of other Coleopterafamilies (JUANet aI. 1991; ROZEC1992; ROZECandRUDEK 1992). On the other hand, extra heterochro-matic segments have also been detected in Bubasbison (COLOMBAet aI. 1996).

Intra- and interspecific CH heterogeneity were ob-served after using fluorochrome staining in all thespecies. In P. (Phytalus) vestita the positive labelingin response to the CMA3 and DAPI indicates that theheterochromatin does not present a specific riches interros of AT or GC base composition (CH with nospecific abundance). Among grasshoppers there arealso reports of species that respond positively tostains with different specificity, such as Arcypterafusca and A. tornosi, whose chromosomes are labeledwith antagonist stains when they have been pre-treated for C-banding (BELLAand GOSÁLVES1991).According to these authors, this seems to oceur dueto protein removal during pretreatment. However, inP. (Phytalus) vestita the fíuorescence pattem wasobtained in chromosomes that had not been pre-treated for C-banding, supporting the suggestion thatthe CH is heterogeneous. In contrast, P. (Phyl-lophaga) aff capillata presented a predominantly neu-tral CH for the fiuorochromes used, only a smallauto some and the sex bivalent showed CMA3+ label-ing (GC-rich). This was also observed in Macraspisfestiva, in which only a small CMA3+ labeling wasdetected in an sexual pair (BIONE1999). On the otherhand, the abundance of AT base pairs in CH ob-served in all chromosomes of L. fuscus was alsodetected in most of the chromosomes of thescarabeoid Pelidnota pal/idipennis (BIONE1999). De-spite the reduced number of Scarabaeoidea speciesanalyzed with fiuorochromes thus far, there is evi-dence that the CH of this group is quite heteroge-neous, presenting both quantitative and qualitativevariations (BIONE 1999; VITTURI et aI. 1999;COLOMBAet aI. 2000a). On the contrary, to the

Hereditas 138. (2003) Karyotypic characterization of Melolonthinae 203

."i'.

Fig. 2a-f. Giemsa C-banding in Phyllophaga (Phytalus) oestita (a, b) and CB/DAPI inP. (Phyllophaga) ali capillata (e). Triple staining (CMAJI)A/DAPI) in P. (Phytalus)vestita (c, d) and Lyogenys fuscus (g, h) and double staining (CMA3/DA) in P.(Phyllophaga) aff capillata (C). Pachytene (a). metaphase I (b) and diplotenes (c, d) in P.(Phytalus) oestita. Note in (a-b) an almost completely heterochromatic X chromosome.In (c-d) the CH blocks positively labeled with CMA3 and DAPI. Zygotene (e) andpachytene (f) in P. (Phyllophaga) ali capillata. Note in f the CMAt blocks in anautosomal bivalent and in the XY p- Metaphases I (g, h) in L. fuscus, with homogeneousCMA3 staining and positive DAPI staining. The arrows indicate the sex bivalents.Bar = 10 JlIIl.

204 R. de Cássia de Moura et a/o Hereditas 138 (2003)

a

c

9Fig. 3a-h. Silver staining and FISH in two Phyllophaga species and in Lyogenysfuscus. Zygotene(a) and diplotene (b) in P. (Phytalus) testita. Pachytene (c) and metaphase I (d) in P.(Phyllophaga) aff capillata. Pachytene (e) and metaphase I (f) in L. fuscus. Note the NORs in a,c and e (arrow) and the rDNA sites in b, d and f (arrowhead). The detail in g and h shows thelabeling of pericentromeric CH with silver nitrate in P. (Phyllophaga) aff capi/lata and L. fuscus,respectively. Observe the argyrophilic behavior of the sex bivalent in metaphase I (g) and indiakinesis (h). Bar = 10 11m.

Hereditas 138 (2003) Karyotypic characterization of Melolonthinae 205

family Tenebrionidae which has a large amount ofCH and whose base composition is predominantlyAT-rich (JUAN et aI. 1991; PLOHL et aI. 1993);

According to some investigators (VIRKKl 1983;VIRKKI et aI. 1984), in Coleoptera the NORs arewidely distributed in one autosome pair, as observedin P. (PhyIlophaga) af! capilIata and three otherScarabaeoidea speeies (BIONE 1999; VITTURI et aI.1999: COLOMBA et aI. 2000a). On the other hand, inmost scarabeoids the NOR is present in the sexbivalent (BIONE 1999), as also observed here for P.(Phytalus) vestita and L.fuscus. A sex bivalent deeplystained with silver has been found in various Seara-baeoidea species during different phases of meiosis.This labeling is independent of the presence of NORsin this chromosome pair (BIONE 1999; VITTURI et aI.1999; COLOMBA et al, 2000a). This pattem was alsodetected in all the species and may result from thepresence of non-nucleolar argyrophilic substances inthe lumen of the XyP bivalent. These substances mayfacilitate the parachute configuration of the sex chro-mosomes and probably play an important role in thesegregation of these chromosomes (VIRKKI et ai.1990, 1991).

Silver nitrate staining showed a preliminary label-ing of the NORs in the three species analyzed. How-ever, only the FISH permitted a precise identificationof the NORs in these species. The present FISHstudy unequivoeally assigned the 25S and 18S (major)rONA loei on a single autosome pair in P. (Phyl-lophaga) aff capillata and X chromosome in P. (Phy-talus) vestita and L. fuscus. On the other hand, littleis known about the ehromosomal loeation of rONAgenes in representatives of the superfamily Seara-baeoidea (VITTURI et aI. 1999; COLOMBA et aI.2000a,b) in eomparison with other Coleoptera groups(PETITPIERRE 1996; GALIAN et ai. 1995; SANCHEZ-GÉA et aI. 2000) in the whieh data have beenpublished.

The heteroehromatie regions of the three speciesstudied were deeply stained with silver, This argy-rophilic behavior of CH has been observed in repre-sentatives of Searabaeoidea and does not depend onthe base eomposition of CH, also occurring in speciessueh as Bubas bison and Pelidnota pallidipennis,whose CH is AT-rieh (COLOMBA et aI. 1996; BIONE1999), Thorectes intermedius, Gymnopleurus sturmiand Geniates borelli, whose CH is in GC-rich (BIONE1999; VITTURI et al 1999; COLOMBA et al 2000a),and in Macraspis festiva, whose CH is neutra1 (BroNE1999).

Although the species studied in the present workhad a 2n = 20,Xyp karyotype, eonsidered to be morefrequent for Melolonthinae, the methods used per-mitted us to eharacterize karyotypie differences be-

tween the two PhyIlophaga species and Lyogenysfuscus in terms of ehromosome Y, NOR localization,distribution of rONA sites and base eomposition ofCH, suggesting that the genomes of the two generaevolved in a different manner.

Acknowledgements - The authors are grateful to Dr. SérgioIde, Instituto Biológico, São Paulo, and Dr. Miguel AngélMorón, Instituto de Ecologia, Mexico, for the taxonomicidentification of the species analyzed in the present study,to Dr. Marcelo Guerra, Departamento de Botânica, UFPE,for permitting the use of the infrastructure for the execu-tion of the FISH technique, to Dr. Neide Santos for acritical review of the manuscript, and to Francisca Tavaresde Lira for technical assistance. Research supported bygrants from Conselho Nacional de Desenvolvimento Cien-tífico e Tecnológico (CNPq) and Fundação de Amparo àCiência e Tecnologia do Estado de Pernambuco(FACEPE).

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