Hereditas 138: 200-206 (2003) Karyotypic characterization of representatives from Melolonthinae (Coleoptera: Scarabaeidae): karyotypic analysis, banding and fluorescent 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, Brasil 2 Departamento de Biologia, Instituto de Ciências Biológicas, Universidade de Pernambuco, UPE, Brasil 3 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 from Melolonthinae (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 three species 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 ali autosomal 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 sex bivalent and one autosomal pair were found to be enriched in GC base pairs with CMA3, and in Lyogenys [uscus CH was positive for DAP!. Silvernitrate staining revealed nucleolar remnants in al1 three species. However, FISH obtained a precise identification of nucleolar organizing regions with an rDNA 18S and 25S probe. A signal of hybridization was seen in each species, being detected in the X chromosome of P. (Phytalus) vestita and Lyogenys fuscus, and in a small autosomal 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: [email protected] The superfamily Scarabaeoidea is represented by 13 and No CERA 1984), G. sterquilinus, Bubas bison families the world over, among them, the Scarabaei- (COLOMBA et al. 1996), Macraspis festiva, Pelidnota dae family is particularly important by having under- pa//idipennis, Lygyrus ebenus, Geniates bore//i (BIONE gone wide adaptive spreading, with a fauna 1999), and Gymnopleurus sturmi (COLOMBA et al. comprising approximately 2000 genera and 25000 2000a). On the other hand, fluorescent in situ hy- species. A total of 362 genera and 4706 species have bridization (FISH) with rDNA probes has been used been recorded in the Neotropical region, and about in a few beetles species (GÁLIAN et al. 1995; DE LA 1777 species belonging to 204 genera have been RÚA et al. 1996; PETITPIERRE 1996; COLOMBAet al. recorded in Brasil (COSTA 2000). 2000a). Although Scarabaeidae represents a rich and diver- The aim of this study is to investigate the meiotic sified fauna, chromosomal studies of this family are chromosomes of males of the species Phyllophaga still scarce. About 323 species have been studied (Phytalus) vestita, Phyllophaga (Phy//ophaga) aff karyotypically thus far, corresponding to 1.29 % of capil/ata and Lyogenys fuscus using conventional the species described (SMITH and VIRKKI 1978; YA· staining, differential chromosome banding techniques DAV and PrLLAI 1979; YADAV et al. 1979; VIDAL and and fluorescent in situ hybridization (FISH). The use NOCERA 1984; COLOMBA et aI. 1996, 2000a; BIONE of these techniques permitted a compara tive analysis 1999). A considerable predominance of a diploid of the three Melolonthinae species studied. number of 2n = 20, biarmed chromosomes and a sex mechanism of the XYp type has been widely detected. MATERIAL ANO METHODS The use of C-banding, silver nitrate staining and base-specific fluorochromes is limited to few The number and sources of the specimens studied scarabeoid species, such as Enema pan (VIDAL and were as follows. Phyllophaga (Phytalus) vestita (10 GIACOMOZZI 1978), Glyphoderus sterquilinus, Eucra- specimens), Phy//ophaga (Phy//ophaga) aff capillata nium arachnoids, Anomiopsoides heteroclyta (VIDAL (12) and Lyogenys fuscus (15) were collected in difTer- l ic' Characlerizalion o' Ki Karyo YP, Sp_PP-01290 IIO\!"\\\ \\111UII\ 1\\\1IU\\ 1\\\1\\111\1\\ \\U\I \\1\1 \\1\\ UI\I \\\\\\\\ CPRTSR-21354-1 \ / Hereditas 138 (2003) Karyotypic characterization of Melolonthinae 201 ent areas of the Atlantic Forest in the State of ASA 25 film, T-MAX 400 ASA film and Kodacolor Pernambuco, northeastern Brazil. 400 ASA film (Kodak), respectively. Male gonads from adult beetles were fixed m Carnoy (ethanol and acetic acid, 3:1) and used to RESULTS obtain meiotic chromosomes. Cytological prepara- tions were obtained by the c1assicalsquashing Conventional staining method and the chromosomes were stained with 2 % Phyllophaga (Phytalus) oestita, Lyogenys fuscus and lactoacetic orcein. C-banding was performed follow- Phyllophaga (Phy/lophaga) aff capillata presented a ing SUMNER (1972), with some slides being stained díploíd number of 2n = 20, meioformula 9Il + XYP' with Giemsa (CBG) and other with DAPl (CBf ín the former ones, beíng 91I + XY p in the P. (Phyl- DAP1). Triple staining with CMA3fDAfDAPl was lophaga) aff capillata, sex-determining mechanisms of performed according to SCHWEIZERet a!. (1983) and the parachute type and an achiasmatic association double staining with CM'A3fDA and DAPlfDA was between the sex chromosomes. All the three species also applied in the threespecies. Silver nitrate stain- have symmetrical karyotypes and apredominance of ing (AgN03) was performed by the method of RUFAS apparently biarmed chromosomes, as observed in et aI. (1987). ln the fluorescent in situ hybridization metaphases I (Fig. Ia-c). The male sex chromosomes (FISH) we used l8S and 25S probes (rDNA) of are represented by a heteromorphic pai r, which form Arabidopsis tha/iana (UNFRIED et a!. 1989; UNFRIED a typical parachute in P. (Phytalus) uestita (Fig. Ia) and GRUNDLER 1990) and thetechnique of and L. fuscus (Fig. 1c), with a very small X chromo- MOSCONE et aI. (1996). The probes were labeled with some and a punctiform Y chromosome (Xy.). In P. bio-ll-dUTP bynick .translation (Life Technologies) (Phyllophaga) aff capillata the sex chromosomes and detected by rat antibiotin antibodies (Dakopatts (XY p) are small and present identical size (Fig. 1b). M0743, DAKO) and anti-rat antibodies (Dakopatts R0270, DAKO) produced with a rabbit TRITC (te- C-banding and fluorochrome staining tramethyl-rhodamine isothiocyanate) conjugate. The The localization of constitutive heterochromatin preparations were counterstained with DAPI (2 ~gf (CH) blocks was defined by C-banding only in P. ml) and mounted with Vectashield H-IOO(} (Vector). (Phytalus) vestira, which presented pericentrorneric Slides were examined with a Leitz Orthoplan pho- blocks in all autosomes. This pattern permitted the tomicroscope. Conventional staining, fluorescence identification of the occurrence of biarmed chromo- and FISH were photographed with Kodak Imagelink somes in this species. The sex bivalent was almost \ • \. , a b 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 parachute configuration 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. biarmed autosomes and a sex mechanism of the (Phyllaphaga) aff capillata (Fig. 2e) slides pretreated parachute type (Xyp), some chromosome rearrange- by C-banding and stained with DAPI (CB-DAPI) ments have led to changes in chromosome morphol- showed the presenee of apparently smaller CH blocks ogy, size and diploid numbers. Some examples are compared to P. (Phytalus) vestira. Triple staining Apogonia sp with 2n = 21,XO (SAHA1973), Apogonia nigricans Apogonia = with CMA3/DA/DAPI in P. (Phytalus) oestita did and sp with 2n 19,XO (MANNA not reveal any differential amount of CH and showed and LAHIRI 1972; YADAV and PILLAI 1974) and that all blocks were labeled both by CMA3+ and Ophthalmoserica karafutoensis with 2n = 18 (KUDOH DAPI + (Fig. 2c-d). However, DAPI labeling was et aI. 1973). more intense than CMA3. In P. (PhylIophaga) aff Although C-bands are localized in the pericen- tromeric region of most of the Scarabaeidae species capillata double staining with CMA3/DA showed the analyzed (BIONE 1999; COLOMBAet aI. 2000a), as presence of CMA3+ blocks only in the sex bivalent and in a small autosomal bivalent (Fig. 2f). No also observed here in P. (Phytalus) vestita and L. DAPI + blocks were detected in this species (result fuscus, other scarabeoids such as Enema pan (VIDAL not shown). On the other hand, the CH of the entire and GIACOMOZZI1978), Glynoderus sterquilinus and chromosome complement in L. fuscus showed homo- Eucranium arachnoides (VIDAL and No CERA 1984) show interstitia1 and telomeric C-bands. This has a1so geneous CMA3 staining (Fig. 2g) and strongly posi- tive DAPI staining (Fig. 2h), clearly