Coleoptera: Scarabaeidae): Karyotypic Analysis, Banding and Fluorescent in Situ Hybridization (FISH

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-

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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 preparations 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

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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 pericentromeric 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 positive DAPI staining (Fig. 2h), clearly showing that the been reported for representatives of other Coleoptera CH is AT-rich. families (JUANet aI. 1991; ROZEC 1992; ROZECand RUDEK 1992). On the other hand, extra heterochro- AgN03 staining and fiuorescent in situ hybridization matic segments have also been detected in Bubas (FISH) bison (COLOMBAet aI. 1996). Silver nitrate staining showed the presence of nucle- Intra- and interspecific CH heterogeneity were observed after using fluorochrome staining in all the olar remnants in only one chromosome pair of each species. In P. (Phytalus) vestita the positive labeling species. In P. (Phy//ophaga) af! capillata the active in response to the CMA and DAPI indicates that the NOR was observed in an autosome (Fig. 3c), in 3 heterochromatin does not present a specific riches in contrast to P. (Phytalus) vestita (Fig. 3a) and L. terros of AT or GC base composition (CH with no fuscus (Fig. 3e) in which the NORs were located in specific abundance). Among grasshoppers there are the sex bivalents. The sex bivalent was strongly also reports of species that respond positively to stained by silver and continued to be labe1ed during stains with different specificity, such as Arcyptera the different phases of meiosis, as can be seen in P. fusca and A. tornosi, whose chromosomes are labeled (Phyllophaga) aff capillata and L. fuscus (Fig. 3g-h). with antagonist stains when they have been pre- Silver nitrate also 1abe1edthe CH of the autosomal treated for C-banding (BELLAand GOSÁLVES1991). biva1ents (Fig. 3g-h). In meiotic bivalents FISH with According to these authors, this seems to oceur due the 18S and 25S ribosoma1 probe (rDNA) only to protein removal during pretreatment. However, in showed one signal of hybridization in each species P. (Phytalus) vestita the fíuorescence pattem was (Fig. 3b,d,f), confirroing the AgN03 staining. Never- obtained in chromosomes that had not been pre- theless, in P. (Phy//ophaga) af! capillata (Fig. 3d), treated for C-banding, supporting the suggestion that labeling oceurred in a small-sized autosome, whereas the CH is heterogeneous. In contrast, P. (Phyl- in P. (Phytalus) vestita (Fig. 3b) and L. fuscus (Fig. lophaga) aff capillata presented a predominantly neu- 3f) the hybridization signals were present in the X tral CH for the fiuorochromes used, only a small chromosome. auto some and the sex bivalent showed CMA3+ labeling (GC-rich). This was also observed in Macraspis DISCUSSION festiva, in which only a small CMA3+ labeling was detected in an sexual pair (BIONE1999). On the other The karyotypic constitution of 2n = 20,Xyp in males hand, the abundance of AT base pairs in CH ob- (meioforroula 9II + Xy.) in Phyllophaga (Phytalus) served in all chromosomes of L. fuscus was also vestita, Phy/lophaga (Phly/lophaga) aff capillata and detected in most of the chromosomes of the Lyogenys fuscus corresponds to the karyotype most scarabeoid Pelidnota pal/idipennis (BIONE1999). De- frequently encountered in the fami1y Scarabaeidae. spite the reduced number of Scarabaeoidea species This karyotype has also been detected in more than analyzed with fiuorochromes thus far, there is evi- 50 % of the Melolonthinae species analyzed thus far dence that the CH of this group is quite heteroge- (SMITH and VIRK.K.l1978; YADAVand PILLAI 1976 neous, presenting both quantitative and qualitative 1979). Although this subfamily is considered to be variations (BIONE 1999; VITTURI et aI. 1999; chromosomally conserved, generally presenting COLOMBAet aI. 2000a). On the contrary, to the Hereditas 138. (2003) Karyotypic characterization of Melolonthinae 203

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Fig. 2a-f. Giemsa C-banding in Phyllophaga (Phytalus) oestita (a, b) and CB/DAPI in P. (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) and pachytene (f) in P. (Phyllophaga) ali capillata. Note in f the CMAt blocks in an autosomal bivalent and in the XY p- Metaphases I (g, h) in L. fuscus, with homogeneous CMA3 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)

9 Fig. 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 the labeling 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 in diakinesis (h). Bar = 10 11m. Hereditas 138 (2003) Karyotypic characterization of Melolonthinae 205 family Tenebrionidae which has a large amount of tween the two PhyIlophaga species and Lyogenys CH and whose base composition is predominantly fuscus in terms of ehromosome Y, NOR localization, AT-rich (JUAN et aI. 1991; PLOHL et aI. 1993); distribution of rONA sites and base eomposition of According to some investigators (VIRKKl 1983; CH, suggesting that the genomes of the two genera VIRKKI et aI. 1984), in Coleoptera the NORs are evolved in a different manner. widely distributed in one autosome pair, as observed in P. (PhyIlophaga) af! capilIata and three other Acknowledgements - The authors are grateful to Dr. Sérgio Scarabaeoidea speeies (BIONE 1999; VITTURI et aI. Ide, Instituto Biológico, São Paulo, and Dr. Miguel Angél 1999: COLOMBA et aI. 2000a). On the other hand, in Morón, Instituto de Ecologia, Mexico, for the taxonomic most scarabeoids the NOR is present in the sex identification of the species analyzed in the present study, bivalent (BIONE 1999), as also observed here for P. to Dr. Marcelo Guerra, Departamento de Botânica, UFPE, for permitting the use of the infrastructure for the execu- (Phytalus) vestita and L.fuscus. A sex bivalent deeply tion of the FISH technique, to Dr. Neide Santos for a stained with silver has been found in various Seara- critical review of the manuscript, and to Francisca Tavares baeoidea species during different phases of meiosis. de Lira for technical assistance. Research supported by This labeling is independent of the presence of NORs grants from Conselho Nacional de Desenvolvimento Cien- in this chromosome pair (BIONE 1999; VITTURI et aI. tífico e Tecnológico (CNPq) and Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco 1999; COLOMBA et al, 2000a). 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206 R. de Cássia de Moura et ai. Hereditas 138 (2003)

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