Hereditas 133: 147- 157 (2000)

Cytogenetic analysis of Epicauta atomaria (Meloidae) and Palembus dermestoides (Tenebrionidae) with Xy sex determination system using standard staining, C-bands, NOk and synaptonemal complex microspreading techniques MARA CRISTINA DE ALMEIDA’, ADILSON ARIZA ZACARO’ and DORALICE MARIA CELLA3 Departamento de Biologia Geral, Setor de Citncias Biolhgicas e da Saude, Universidade Estadual de Ponta Grossa, UEPG, Brazil Departamento de Biologiu Geral, CCBS, Universidade Federal de Vigosa UFV Vigosa, MG, Brazil Departamento de Biologia, Instituto de Biocitncius, Universidade Estadual Paulista, UNESP, Rio Clara, SP, Brazil

Almeida, M. C. de, Zacaro, A. A. and Cella, D. M. 2000. Cytogenetic analysis of Epicauta atomaria (Meloidae) and Palembus dermestoides (Tenebrionidae) with Xy, sex determination system using standard staining, C-bands, NOR and synaptonemal complex microspreading techniques-Hereditas 133: 147- 157. Lund, Sweden. ISSN 0018-0661. Received September 20, 2000. Accepted December 13, 2000

The mitotic and meiotic chromosomes of the Epicautu atomaria (Meloidae) and Palembus dermestoides (Tenebri- onidae) were analysed using standard staining, C-banding and silver impregnation techniques. We determine the diploid and haploid chromosome numbers, the sex determination system and describe the chromosomal morphology, the C-banding pattern and the chromosome(s) bearing NORs (nucleolar organizer regions). Both species shown 2n = 20 chromosomes, the chromosomal meioformula 9 + Xy,, and regular chromosome segregation during anaphases I and 11. The chromosomes of E. utomariu are basically metacentric or submetacentric and P. dermestoides chromosomes are submetacentric or subtelocentric. In both beetles the constitutive heterochromatin is located in the pericentromeric region in all autosomes and in the X, chromosome; additional C-bands were observed in telomeric region of the short arm in some autosomes in P. dermestoides. The yp chromosome did not show typical C-bands in these species. As for the synaptonemal complex, the nucleolar material is associated to the 7th bivalent in E. aromaria and 3rd and 7th bivalents in P. dermestoides. Strong silver impregnated material was observed in association with Xy, in light and electron microscopy preparations in these species and this material was interpreted to be related to nucleolar material. Key words: kuryotype, chromosome, heterochromatin, pachytene, meiosis, nucleolus, polyphaga, coleoptera.

Mura Cristina de Almeida, Departamento de Biologia Geral, Universidade Estadual de Ponta Grossa, UEPG, Setor de Ciincius Bioldgicas e du Saude, Av. Carlos Cavalcanti, n.4748, CEP: 84030000, Uvarunus, Ponta Grossu, Paruna, Brazil. E-mail: [email protected]

The order Coleoptera has approximately 350,000 that could explain its widespread presence in a great known species; the total number may be ten times number of coleopterans. higher. The families Meloidae and Tenebrionidae During meiosis, the association between the X and comprise, respectively, about 1% and 6% of this order y sex chromosomes may vary depending on the spe- (COSTA 1999). According to SMITH and VIRKKI cies and on the chromosome differentiation during (1978) and JUANand PETITPIERRE(1 989), 24 species past evolution. This association can be established by of Meloidae and 200 of Tenebrionidae have well nucleolar material and/or by achiasmatic synapsis characterized karyotypes, i.e. the diploid number, the (SMITH and VIRKKI 1978; JUANet al. 1993; PETIT- chromosome morphology and the type of the sex PIERRE 1996). chromosome determination system are known. Until the sixties, all cytogenetic analyses on As postulated by SMITH and VIRKKI (1978), the Coleoptera were made using sectioning or “squash” great majority of the cytogenetically described techniques. These techniques showed great karyotype coleopteran species possess the basic chromosome uniformity in the majority of the studied species. meioformula 9 + Xy,, which is considered primitive However, at the end of the sixties, new techniques for this group. The “p” represents an special meiotic arose shedding light to longitudinal differentiation of configuration of the associated sex chromosomes the chromosomes estabhhing the banding tech- which resembles a parachute during metaphase I. niques. The presence and the identification of specific According to VIRKKI (1984), the sex determination regions on mitotic and meiotic Coleoptera chromo- system of the type Xy, can not represent a primitive somes, such as constitutive heterochromatin (C-band- condition, but it could have an adaptive advantage ing pattern) and nucleolar organizer regions (NORs) 148 Mara Cristina et al. Hereditas 133 (2000) allowed a better characterization of each kary- hypotonic solution (tap water, for 3 min) and fixed in otype. They shed information on the level of struc- Carnoy I (methanol: glacial acetic acid, 3:l parts by tural differentiation of the chromosomes and volume, for at least 30 min). Each fixed testis was facilitated the comparison of karyotype of related transferred into a drop of acetic acid solution (45%), species. It was shown that during meiosis the associa- that was placed on the surface of the slide and then tion of sex chromosomes could be established by the material was macerated to form a cellular suspen- heterochromatic regions or by NORs. sion. The slides were dried at 35-40°C (heating metal The study of heterochromatin and NORs in the plate). Standard staining was accomplished using chromosomes in a few number of species of Giemsa (3% of Merck commercial solution in phos- Coleoptera was reported giving information mainly phate buffer at pH 6.8, for 12-15 min), rinsed briefly about karyotype differentiation among species that with distilled water, and air-dried. C-banding and possess the basic chromosome meioformula for the NOR silver impregnation were performed according group (9 + Xy,). As examples we can cite Coccinelli- to the methodologies described by SUMNER(1990) dae (MAFFEIet al. 2000), Curculionidae (VIRKKIet and HOWELLand BLACK(1980), respectively. Light al. 1991), Carabidae (Rozek and Maryanska-Nada- microscopy (LM) for routine cytological analyses and chowska, 1991), Chrysomelidae (PETITPIERRE1996), photomicrography were carried out using a Zeiss Hydrophilidae (ANGUS1983), Tenebrionidae (JUAN photomicroscopy. For photomicrography it was em- et al. 1993; PETITPIERREet al. 1995) and Cicindelidae ployed an Agfa-Gevaert Copex Pan A.H.U. film. (GALIANet al. 1995). Synaptonemal complex (SC) microspreading analy- In general, the constitutive heterochromatin in the ses were accomplished according to the methodology chromosomes of these beetles is found in the pericen- described by LOIDLand JONES(1986). The analyses tromeric region. Some species may show additional of silver impregnated (HOWELLand BLACK1980) SC heterochromatic regions (additional C bands) in the preparations were performed using a Zeiss EM9-S2 interstitial and/or telomeric regions. In the sex chro- transmission electron microscopy (TEM) and the mosomes, the constitutive heterochromatin has a electron micrography were made on Kodak 4489 film variable occurrence and can be located in the pericen- plates. tromeric region and/or distributed along the chromo- some according to the level of chromosome differentiation. The pattern of NORs is not well RESULTS established in beetle families since it can be located in Standard staining the autosomic pairs and/or sex chromosomes. When not evident in the autosomes, the visualization of the The spermatogonial metaphase of E. atomaria and P. NORs is restricted to the meiotic cycle and located dermestoides cells showed a diploid number of 2n = between the chromosome X and y (JOHNand LEWIS 20 chromosomes (Fig. 1). The chromosome meiofor- 1960). mula 9 + Xy, was observed in all cells in diakinesis Here we attempt to characterise the karyotype of and metaphase I (Fig. 2a-b, Fig. 2e-f). In all two coleopterans species, Epicauta atomaria metaphases I1 observed, the haploid complement n = (Meloidae) and Palembus dermestoides (Tenebrion- 9 + X, or n = 9 fy, was evidenced, indicating that idae). We analyse the mitotic and meiotic chromo- chromosomes segregate normally during anaphase I somes preparations to describe the diploid number, (Fig. 2c-d, Fig. 2g-h). The karyotype of E. atomaria the chromosome morphology, the type of sex chro- spermatogonial metaphases shows that the diploid mosome determination system, the C-banding pat- complement is formed by metacentric and submeta- tern, and the chromosomes bearing NORs. centric chromosomes while the P. dermestoides diploid complement is formed by submetacentric and subtelocentric chromosomes. The X, chromosome of MATERIAL AND METHODS both species is metacentric of medium size and usu- We used 11 E. atomaria and 65 P. dermestoides adult ally is difficult to be identified due its similarity with males. The E. atomaria beetles were collected at the autosomal chromosomes of the same size. The yp is Instituto de Biocikncias Experimental Garden, UNESP metacentric and is characterized as the smallest chro- (Rio Claro, SP, Brazil) and at the Horto Florestal mosome of the karyotype in both species (Fig. 1). Navarro de Andrade (Rio Claro, SP, Brazil), and the In both beetles it was observed in the mitotic P. dermestoides specimens were obtained from do- metaphase and metaphase 11, that negative heteropy- mestic strains from Limeira (SP, Brazil). cnosis appears in the pericentromeric region, in the The chromosome preparations were obtained from majority of the chromosomes, and in the short arms adult male testes. The material was immersed in of some chromosomes (Fig. 1, Fig. 2c-d, Fig. 2g-h). Hereditas 133 (2000) Cytogenetic analysis of Epicauta atomaria and Palembus dermestoides 149

Additionally, it was observed that in some of these telomeric region of the short arm of some chromo- cells the y, chromosome possesses negative somes (Fig. 3d). heteropycnosis. In the metaphase I cells of both species, the peri- In diakinesis cells it was noted the occurrence of centromeric C-bands extend along the short arm in one chiasma per autosomal bivalent and that the X, almost all the autosomes and in the X, sex chromo- and yp chromosomes are associated by the parachute some (Fig. 4). In the metaphase I cells of E. atomaria configuration (Fig. 2a, Fig. 2e). Furthermore, the yp the y, chromosome shows a tenuous C-band (Fig. chromosome showed negative heteropycnosis in all 4a-b) while no distinguishable band was observed in analysed diakinesis and metaphase I cells (Fig. 2a-b, the P. dermestoides yp chromosome at the same mei- Fig. 2e-f). otic phase (Fig. 4c-d). Analyses of metaphase I1 have evidenced similar C-banding pattern to that one C-banding noted in the metaphase I cells (Fig. 5). The E. atomaria and P. dermestoides spermatogonial Silver nitrate impregnation metaphases showed that the constitutive heterochro- matin of the autosomes and X, chromosome is lo- LM of testicular chromosome preparations of E. cated in the pericentromeric region (Fig. 3). The E. atomaria and P. dermestoides, which were previously atomaria y, shows a tenuous C-band probably in the analysed with conventional staining and then submit- pericentromeric region (Fig. 3b) and the P. der- ted to silver impregnation, did not show the typical mestoides yp does not exhibit any C-band (Fig. 3d). NOR silver impregnation in the spermatogonial In both species, the pericentromeric C-band extends metaphase chromosomes. towards to the short arm. In P. dermestoides addi- On the other hand, TEM analysis of E. atomaria tional heterochromatin was also detected in the pachytenic cells showed that the nucleolar material is

Fig. la and b. Karyotypes of Epicauta atomaria a and Palembus dermestoides b spermatogonial metaphases (2n = 18 + Xy,) standard staining with Giemsa. The chromosomes are metacentric, submetacentric, and subtelocentric. Bar = 5 pm. 150 Mara Cristina et al. Hereditas 133 (2000’,

Fig. 2a-h. Epicauta atomaria a-d and Palembus dermestoides e-h male meiotic chromosomes analysed by standard staining. Diakinesis a and metaphase I h showing the meioformula 9 + Xy, and the negative heteropycnotic y, chromosome; metaphases I1 with n = 9 + X, c and n = 9 + y, d. Diakinesis e and metaphase I f evidencing the meioformula 9 + Xy, and also the negative heteropycnotic y, chromosome; metaphases I1 with n = 9 + X, g and n = 9 + y, h. Arrows indicate chiasmata. Bar = 5 pm.

associated to the 7th autosomal bivalent (Fig. 6a). In to 3rd and 7th autosomal bivalents (Fig. 6b). Addi- P. dermestoides, the nucleolar material is associated tionally, in both species strong silver impregnation Hereditas 133 (2000) Cytogenetic unalysis of Epicautu atomaria and Palembus dermestoides 151 was detected in association to Xy, sexual bivalent morphological variability observed in the E. atomaria and it was interpreted as being related to nucleolar and P. dermestoides chromosomes seems to show that material (Fig. 6). there have been structural rearrangements such as Light microscopy showed strong silver impregna- pericentric inversions or alterations in the constitutive tion of nucleolar material only in the Xy, sexual heterochromatin content. bivalent in all cells in diakinesis and metaphase I Some karyotypic characteristics observed in E. (Fig. 7), in both species. LM analysis of metaphase I1 atornuria, such as the diploid chromosome number microspreading E. atornuria cells showed nucleolar and the sex chromosome system agree with the ones material associated to only one chromosome of the described for others species of the same genera, for haploid complement (Fig. 8). example, Epicauta anthracina, Epicauta cinerea, Epi- cuuta isthmica, Epicauta murina, Epicauta pennsyl- vanica, Epicauta rufJipedes and Epicauta n.sp (SMITH DISCUSSION and VIRKKI1978; FERREIRAand MESA 1977). Chro- Recently, some authors have employed more refined mosome morphology has been described only for E. techniques to analyse, to characterise, and to better anthracina, in which all chromosomes were classified differentiate the chromosomes of the beetles that as metacentric (FERREIRAand MESA 1977). have the basic chromosome meioformula 9 + Xy, Positive heteropycnosis in the sex chromosomes (ANGUS1983; POSTIGLIONIet al. 1991; VIRKKIet al. during the meiosis is observed in the majority of 1991; UGARKOVICet al. 1994; PETITPIERRE1996). beetles; this heteropycnosis seems, however, to vary The standard staining analysis of the karyotypes among related species (FERREIRAand MESA 1977; and the behaviour of the E. atomaria and P. der- YADAVet al. 1985; JUANet al. 1993). Positive het- mestoides meiotic bivalents show the general eropycnosis was also observed in X, and y, sex coleopteran basic cytogenetic characteristics. The chromosomes of Meloidae (SMITHand VIRKKI 1978) chromosomal morphology differs between these spe- and Tenebrionidae (BISOIand PATNAIK1988; JUAN cies, except in that of the y, sex chromosome. The et al. 1993).

Fig. 3a-d. Epicauta atomaria a-b and Palembus dermestoides c-d spermatogonial metaphases submitted to both standard staining a and c and C-banding b and d. The large and the small arrows indicate pericentromeric and telomeric C band, respectively. Bar = 5 pm. 152 Mara Ciaistinu et al. Hereditas 133 (2000)

Fig. 4a-d. Epicauta atomaria a-b and Palembus dermestoides c-d metaphases I submitted to both standard staining a and c and C-banding b and d. Small and large arrows indicate respectively C band and Xy, bivalent. Bar = 5 pm.

The heteropycnotic pattern observed in the E. large, and G. rusticum, in which the X, is very small atomaria and P. dermestoides X, and yp chromo- (JUANand PETITPIERRE1990). somes, i.e. X, allocyclic and yp negative heteropyc- The pericentromeric C-banding pattern noted in notic, differs of the positive heteropycnotic pattern the E. atomaria and P. dermestoides mitotic and found in these chromosomes of other Meloidae spe- meiotic chromosomes agrees with that described for cies (E. rujipedes, E. isthmica, E. grammica, Mylabris most Coleoptera species (ANGUS1983; JUANet al. thunbergi, Panyculolytta and Tetraonyx frontalis) 1993; UGARKOVICet al. 1994). Additional C bands (SMITHand VIRKKI1978; BISOI and PATNAIK1988) were seen in the telomeric region of the short arm in and Tenebrionidae (Hoplobrachiurn dentipes, Pachyc- some chromosomes of P. dermestoides. Telomeric era?coromandelensis and Misolampus goudoti) (BISOI C-bands were also detected in chromosomes of and PATNAIK1988; JUANet al. 1993). The difference Misolampus goudoti (Tenebrionidae) by JUAN et al. involving heteropycnosis can be due to the chromo- (1993). These additional C-bands have probably some condensation and/or the presence of special arisen by means of small tandem duplications as type of chromatin. Besides, the difference in size of proposed by KING and JOHN (1980) when they the X and y sex chromosomes among species of the analysed the C-banding pattern of some species of same family or of the same genera is very common in Orthoptera. Coleoptera, due to the occurrence of independent In both beetles, the y, chromosome does not process of differentiation. The Meloids Epicauta n.sp. present a particular heterochromatic marking. The and Pyrota decorata have extremely large X and y C-banding technique does not show all types of the chromosomes when compared to the ones found in E. heterochromatin (SUMNER1990), so that we can not isthmica and Paniculolytta sanguineoguttata (SMITH exclude the possibility of the yp chromosome being and VIRKKI 1978). Difference in size between sex heterochromatic. chromosomes also occur in Tenebrionidae, for exam- The employment of more refined techniques, such ple, Gonocephalum patrueh, in which the X, is very as in situ hybridization using satellite DNA probes, Hereditas 133 (2000) Cytogenetic analysis of Epicauta atomaria and Palembus dermestoides 153 will certainly provide information about the nature of the X, and, also, along the y, chromosome of the two the chromatin in the y, chomosome. JUAN et al. unrelated beetles. In these phases, the C-band is (1993) have confirmed the presence of repetitive totally or partially coincident with negative heteropy- DNA in the y, chromosome of coleopterans species cnosis in the majority of the chromosomes, but not in using fluorescent in situ hybridization and the yp, showing that this chromosome may contain a fluorochrome staining. different type of chromatin. Even through the two beetles that we have studied The analysis of the C-banding pattern obtained for share the same basic karyotype, they are different at the Xy, bivalent in the metaphase I of both species the C-band level. allowed to indicate that the parachute configuration In the spermatogonial metaphases, diakinesis and is not assembled by a constitutive heterochromatic the metaphases I and 11, negative heteropycnosis association. appears in the pericentromeric region and along the Although the C-banding technique does not evi- short arms in almost all autosomal chromosomes, in dence all the particular types of chromatin, the em-

Fig. 5a-f. Epicauta atomaria a-d and Palembus devmestoides e-f metaphases I1 with X, a, b, e and f) and yp chromosomes c-d. a and c Standard staining. b and d The same cells as in a and c submitted to C-banding. e Standard staining. f The same cell as in e subjected to C-banding. Arrows indicate pericentromeric C band. Bar = 5 pm. 154 Mara Cristina et al. Hereditas 133 (2000)

Fig. 6 a and b. Epicauta atomaria a and Palembus dermestoides b ultra-structural karyotypes of pachytenic nucleus, meiofomula 9 + Xy,, analysed by silver nitrate impregnation. N = nucleolar material. Bar = 10 pm. ployment of this technique has shown that the Silver nitrate impregnation of E. atomaria and P. X, and y, chromosomes are totally differentiated dermestoides spermatogonial metaphases did not in some Coccinelinae and Tenebrionidae species, show NORs. As postulated by GOODPASTUREand being associated by heterochromatic regions (DRETS BLOOM (1975) the absence of NOR(s) in mitotic et al. 1983; JUAN et al. 1993; UGARKOVICet al. chromosomes can be due to a circumstance that 1994). ribosome cistrons are disposed in small groups in the Hereditas 133 (2000) Cytogenetic analysis of Epicauta atomaria and Palembus dermestoides 155 chromosomes and, consequently, the proteins respon- cistrons in the Xyp bivalent could be confirmed only sible to silver nitrate precipitation could be small so using rDNA probes as reported by JUAN et al. that NOR(s) are not seen. Nevertheless, nucleolar (1993). material was observed associated to the E. atomaria The occurrence of nucleolar material associated to 7th and Xy, bivalents and P. dermestoides 3rd, 7th Xy, sex bivalent and, in some cases, also associated and Xy, bivalents, which probably remains associ- to autosomal bivalents, was observed in Scarabaeidae ated to the bivalents in the meiotic division for longer (SMITH and VIRKKI 1978), in Dermestidae (JOHN than that one associated to the chromosomes in the and SHAW1967), and in Tenebrionidae (JUANet al. mitotic division. 1993). In CicindeIidae (Adephaga), which have the The presence of nucleolar material in the Xy, male XXXy type of sex determining system, the bivalents of E. atomaria and P. dermestoides does presence of a NOR associated to only one X chromo- not mean that ribosome cistrons are located in this some and to y chromosome was evidenced. In two bivalent, but the nucleolar material could be other species of the same family, which show the XO transferred to Xy, bivalent during the early pro- sex determining system, it was observed the occur- phase contributing for regular association and segre- rence of NOR associated to an autosomal bivalent gation during meiosis. The occurrence of ribosome and not to the X chromosome (GALIANet al. 1995).

Fig. I a-d. Epicauta atomaria a-b and Palembus devmestoides c-d diakinesis, meioformula 9 + Xy,, submitted to both standard staining a and c and silver nitrate impregnation b and d. The Xy, is deeply impregnated by silver nitrate (arrows). Bar = 5 pm. 156 Mara Cristina et al. Hereditas 133 (2000)

Estado de SZo Paulo) for the financial support. The authors are also grateful to Dr Eduard Petitpierre, Universitat de les Illes Balears, Spain, and Dr Anssi Saura, Umei Univer- sity, Sweden, for manuscript suggestions and critical read- ing.

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