- Email: [email protected]
Karyotype, banding and rDNA FISH in the scarab beetle Anoplotrupes stercorosus (Coleoptera Scarabaeoidea: Geotrupidae). Description and comparative analysis
Micron 35 (2004) 717–720 www.elsevier.com/locate/micron
Karyotype, banding and rDNA FISH in the scarab beetle Anoplotrupes stercorosus (Coleoptera Scarabaeoidea: Geotrupidae). Description and comparative analysis Mariastella Colombaa,*, Roberto Vitturib, Nicola Volpeb, Antonella Lanninob, Mario Zuninoa a
Istituto di Ecologia e Biologia Ambientale, Universita` di Urbino “Carlo Bo”, via Sasso 75, 61029 Urbino (PU), Italy b Dipartimento di Biologia Animale, Universita` di Palermo, Via Archirafi 18, 90123 Palermo, Italy
Abstract Six specimens of Anoplotrupes stercorosus (Coleoptera Scarabaeoidea: Geotrupidae) were analysed using conventional staining, banding techniques and fluorescent in situ hybridization with a ribosomal probe (rDNA FISH). Detailed karyotype description was also joined to a comparative analysis between present data and those previously reported for Thorectes intermedius [Chromosome Res. 7 (1999) 1]. The two species, both belonging to the tribe Geotrupini, show the same modal number but different autosomal morphology which is in contrast with the high chromosome stability argued for Geotrupinae during the last three decades. Moreover, a detailed comparison reveals the occurrence of a plesiomorphic condition in A. stercorosus with respect to the apomorphic one of T. intermedius. This finding agrees with phylogenetic relationships proposed for the two genera based on morphological and anatomical characters. q 2004 Elsevier Ltd. All rights reserved. Keywords: Coleoptera Scarabaeoidea Geotrupidae; Cytogenetics; 18S rDNA FISH
1. Introduction The coleopteran superfamily Scarabaeoidea, whose origins can be traced back to the late Mesozoic (200 MYA), is a cosmopolitan, monophyletic group which includes about 35,000 described species. Up to the present time, this taxon has been investigated from many different points of view, as demonstrated by the high number of studies (i.e. Halffter and Matthews, 1966; Moro´n-Rios, 1984; Hanski and Cambefort, 1991 and authors quoted therein) reporting on phylogeny, ecology, biogeography, behaviour and reproductive biology of these interesting organisms. On the contrary, other subjects including, among others, immunology, molecular biology and cytogenetics remain mostly unknown. Available karyological data are limited to the haploid and/or diploid numbers of about 200 species, the karyotype description of some of them (Salamanna, 1965, 1972; Vidal et al., 1977; Yadav et al., 1979, 1989) and chromosome banding (silver staining and/or C-banding) of Enema pan (F.) (Dynastinae) (Vidal and Giacomozzi, 1978) and Bubas bison L. and Glyphoderus sterquilinus (Westwood) (Scarabaeinae) (Colomba et al., 1996). Recently, fluorescent * Corresponding author. Tel.: þ 39-722-303-438; fax: þ39-722-303-436. E-mail address: [email protected] (M. Colomba). 0968-4328/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2004.04.004
in situ hybridization with ribosomal sequences as a probe (rDNA FISH) has mapped major ribosomal clusters (18S-28S rDNA) in three species of three different families, Thorectes intermedius (Costa) (Geotrupidae) (Vitturi et al., 1999), Gymnopleurus sturmi McLeay (Scarabaeidae) (Colomba et al., 2000a) and Dorcus parallelipipedus L. (Lucanidae) (Colomba et al., 2000b). The genus Anoplotrupes (Geotrupidae) was previously described by Jekel (1865) as a subgenus of Geotrupes. According to Zunino (1984), within the tribe Geotrupini, this genus shows a close phylogenetic relationship with the Asiatic genera Glyptogeotrupes Nikolaev and Epigeotrupes Bovo and Zunino. It comprises few species distributed in North America (North of Mexico), with the exception of A. stercorosus (Scriba, 1790) whose distribution covers the European fraction of the Palearctic Region with a frequency gradient decreasing southwards (Zunino, 1984; Martı´n-Piera and Lopez-Colo´n, 2000). In Mediterranean areas, it mainly occurs in semi deciduous or pine woods (at high altitudes). In the present study, we provide an extensive cytogenetic analysis of A. stercorosus using both classical (silver staining, C-banding and fluorochrome staining) and molecular (rDNA FISH) methods. Results are also related with the corresponding ones of another Geotrupini, T. intermedius, previously investigated by Vitturi et al. (1999).
718
M. Colomba et al. / Micron 35 (2004) 717–720
Such a comparison represents an initial contribution to the reconstruction of chromosomal changes that have occurred during the Geotrupidae evolution.
2. Materials and methods Six Anoplotrupes stercorosus (four males and two females), identified according to Zunino (1984); Baraud (1992), were collected at the end of May, 2001 in the beech wood near Busana (Reggio Emilia, North Italy, in the ‘Parco Regionale dell’Alto Appennino Reggiano’). Chromosome preparations were obtained by the airdrying technique after in vivo colchicine (0.15%) treatment. Unfortunately, females did not provide good-quality material for chromosome analysis, therefore, data reported herein resulted from the males. Slides were subjected to Giemsa staining (Vitturi et al., 1996), C-banding (Sumner, 1972), silver staining (Ag-NOR) (Howell and Black, 1980), DAPI and CMA3 staining (Schmid et al., 1983) and rDNA FISH (Colomba et al., 2000b). Chromosomes were classified on the basis of the criteria proposed by Levan et al. (1964). rDNA FISH was carried out on fixed spermatocyte chromosomes using the 18S rDNA of the sea urchin Paracentrotus lividus (Echinodermata) as a probe. Nick translation labelling of the probe with digoxigenin and hybridization experiments were performed according to manufacturer’s (Roche) instructions. Slides were mounted in an antifade solution containing propidium iodide (PI) (5 mg/ml) and viewed under a Leica I3 filter set (BP 450490; LP 515). Microphotographs were taken using a Leica microscope on Kodak Ektacolor 800 ASA film. The systematic scheme adopted in the present paper is that proposed by Lawrence and Newton (1995) for Scarabaeoidea, and that of Zunino (1984) for Geotrupidae.
ranged from about 4.8 to 1.8 mm, showed meta-/submetacentric (M/SM) (pairs no. 3, 4, 5, 6, 8) and subtelo-/acrocentric (ST/A) (pairs no. 1, 2, 7, 9, 10) morphologies. The sex pair consisted of two small elements (M and ST), about 1.4 mm long, whose identification was impossible, since, due to the lack of female chromosome preparations, we have not been able to conclusively distinguish the X from the Y. C-banding showed that heterochromatin coincided with one or both telomeric regions in nearly all chromosomes (18 spreads analysed) (Fig. 2). In a few chromosomes a small heterochromatic dot was located at the centromeric region (Fig. 2, see arrow). Silver staining pattern was repetitive and consisted of argentophilic dots corresponding to heterochromatic regions (28 spermatogonial metaphases analysed) (Fig. 3). A diffuse silver stainability also characterized bivalents at early (Fig. 4A) and late (Fig. 4B) metaphase I stage. Both in mitotic (Fig. 3, see arrows) and meiotic (Fig. 4B, see arrow) spreads, the elements of the sex pair were distinguished by their large silver deposits. CMA3 staining (GC-specific) evidenced two mediumlarge elements—probably the NOR bearing ones—with a bright paracentromeric area (Fig. 5A), whereas DAPI (ATspecific) stained all chromosomes homogeneously (Fig. 5B). rDNA FISH constantly mapped major ribosomal sites in correspondence of the CMA3 positive regions of the two medium-large chromosomes (25 spreads analyzed). Observed hybridized areas significantly varied in dimension, resulting either both large (cytotype A) (10 spreads) (Fig. 6A), or different in size (cytotype B) (15 spreads) (Fig. 6B), thus implying the occurrence of an intercellular variability in the number of ribosomal loci. This finding will not be discussed further since polymorphism in the copy number of rDNA genes is well-known for many animal taxa (Zurita et al., 1977).
4. Discussion 3. Results Giemsa staining of spermatogonial metaphases (32 spreads) of A. stercorosus gave the diploid chromosome number ð2nÞ of 22 which confirms that previously reported by Yadav et al. (1979) for a different strain of the same species (sub Geotrupes). Chromosomes, paired according to their length and centromere position, were arranged into 11 pairs, including ten autosomes and the heteromorphic sex elements (XY) (10 spreads analysed) (Fig. 1). Autosomes whose dimensions
In the last thirty years it has been maintained that karyotypic homogeneity occurs within Geotrupinae (Salamanna, 1972; Yadav and Pillai, 1979). Taking into account that available data on scarab beetles were obtained by dated and non-specific analyses, the present study was carried out to test the reliability of this hypothesis. The diploid number ð2n ¼ 22Þ and the sex-determining mechanism (XX/XY) of A. stercorosus are consistent with those reported for eight Geotrupinae species studied so far (Yadav et al., 1979; Colomba, 1998; Vitturi et al., 1999).
Fig. 1. Karyotype of Anoplotrupes stercorosus obtained from a Giemsa stained spermatogonial metaphase (2n ¼ 22; 10AA þ XY; 11 M/SM þ 11 ST/A). The Fondamental number (FN), on the basis of chromosome morphology described in the results section, is FN ¼ 33 (M/SM chromosomes and ST/A chromosomes are considered as biarmed and monoarmed, respectively).
M. Colomba et al. / Micron 35 (2004) 717–720
Fig. 2. C-banded mitotic chromosomes of A. stercorosus.
Such a homogeneity, evidenced by a low resolution investigation, steadily decreases when a more accurate analysis is performed. In fact, within the tribe Geotrupini, a comparison between A. stercorosus and T. intermedius karyotypes reveals marked differences in chromosome morphology. Apart from the sex elements which show very similar morphology and dimension in both species, autosomes consist of five biarmed and five monoarmed pairs in A. stercorosus, and of 10 monoarmed pairs in T. intermedius. This finding refutes the high stability in chromosome morphology previously argued for the subfamily Geotrupinae (Salamanna, 1972). A lack of a karyological homogeneity within Geotrupinae is largely confirmed when analysis is extended to: 1. NOR number. Major ribosomal clusters are mapped by FISH on one chromosome pair in A. stercorosus and on two chromosome pairs in T. intermedius (Vitturi et al., 1999); 2. heterochromatin location. After C-banding, heterochromatin is centromeric in T. intermedius (Vitturi et al., 1999), whereas it is mostly telomeric in A. stercorosus; 3. heterochromatin base-pair composition. After CMA3 staining, all heterochromatin including both constitutiveand NOR-associated one, is highly compartmentalized in GC base pairs in T. intermedius (Vitturi et al., 1999) whereas, in A. stercorosus, GC-rich base pairs correspond only to NOR-associated heterochromatin. This finding extends the data in previous papers (Colomba et al., 1996, 2000a,b; Vitturi et al., 1999) in which it has been shown that advanced cytogenetic techniques can reveal a high karyological diversification within Scarabaeoidea.
Fig. 3. Silver stained spermatogonial chromosomes of A. stercorosus (arrows indicate the sex chromosomes).
719
Fig. 4. Silver stained meiotic chromosomes of A. stercorosus. Early metaphase I (A); and late metaphase I (B) (arrow indicates the sex bivalent showing large silver deposits).
As already demonstrated for all scarab beetles species so far investigated, in A. stercorosus the silver stainability of all chromosomes was the result of an extensive Ag-positive reaction of heterochromatin. Such non-specific staining prevented unequivocal identification of NORs, so major ribosomal sites were mapped by rDNA FISH. CMA3 staining yielded more information than silver impregnation since, in line with rDNA FISH pattern, it showed NORs on one chromosome pair. The discrepancy between silver and chromomycin patterns is the result of a varying reaction of heterochromatic DNA (entirely silverpositive) to the fluorochrome. In fact, only the NORassociated heterochromatin fluoresced brightly, while the constitutive one reacted homogeneously. This heterogeneous response to CMA3 staining in A. stercorosus disagrees with results obtained in all Scarabaeoidea species analyzed so far, in which both types of heterochromatic DNA react in the same way. In particular, heterochromatin was found to be CMA3-positive in eight species (Colomba, 1998, 2000a; Vitturi et al., 1999), CMA3-negative in 1 species (Colomba, 1998), CMA3-neutral in two species (Colomba, 1998; Colomba et al., 2000b) and DAPI-positive in one species (Colomba et al., 1996). Finally, it is maintained that the occurrence of two NORs and a low level or the absence of chromatin compartmentalization are to be considered as the basic karyotypic form in most vertebrates (Schmid, 1978) and some invertebrates (Thiriot-Quievreux and Insua, 1992; Vitturi et al., 2002). If we extend this assumption to the tribe Geotrupini, A. stercorosus shows primitive karyotipic characters
Fig. 5. Fluorochrome stained spermatogonial metaphase plates of A. stercorosus. (A) CMA3 evidenced a bright paracentromeric area in each element of one chromosomes pair; (B) DAPI stained homogeneously all chromosomes.
720
M. Colomba et al. / Micron 35 (2004) 717–720
Fig. 6. rDNA FISH of A. stercorosus spermatogonial chromosomes. The micrographs were taken with a Leica I3 filter set allowing the simultaneous visualization of the PI-stained chromosomes (red colour) and the hybridization sites of the 18S rDNA probe (yellow colour) Hybridization signals are similar in size (A); and different in size (B) (scale bar: 16 mm ¼ 10 mm).
(two NORs and uncompartmentalized heterochromatin) when compared to T. intermedius (more than two NORs and evident GC-rich heterochromatin). This conclusion agrees with the phylogeny of Geotrupini proposed by Zunino (1984). In this scheme, based on numerous morphological and anatomical characters analyzed according to criteria proposed by Hennig (1996), six genera among which is included Anoplotrupes, represent the sister-group, relatively primitive, of the phyletic branch leading to the genera Thorectes and Trypocopris. Acknowledgements This work was supported by a MURST fund (Fondi di Ricerca Scientifica ex 60%) to M. Colomba. References Baraud, J., 1992. Cole´opte`res Scarabaeoidea d’ Europe, Fe´de´ration Franc¸aise des Sciences Naturelles et Socie´te´ Linne´enne, Lyon. Colomba, M., 1998. L’organizzazione del cariotipo nei Coleotteri Scarabaeoidea: materiali per un’analisi evolutiva. PhD Dissertation. Dipartimento di Biologia Animale. Universita` di Palermo, Palermo, Italy. Colomba, M., Monteresino, E., Vitturi, R., Zunino, M., 1996. Characterization of mitotic chromosomes of the scarab beetles Glyphoderus sterquilinus (Westwood) and Bubas bison (L.) (Coleoptera: Scarabaeidae) using conventional and banding techniques. Biologisches Zentralblatt 115, 58 –70. Colomba, M., Vitturi, R., Zunino, M., 2000a. Karyotype analysis, banding and fluorescent in situ hybridization in the scarab beetle Gymnopleurus sturmi McLeay (Coleoptera Scarabaeoidea: Scarabaeidae). The Journal of Heredity 91(3), 260 –264. Colomba, M., Vitturi, R., Zunino, M., 2000b. Chromosome analysis and rDNA FISH in the stag beetle Dorcus parallelipipedus L. (Coleoptera: Scarabaeoidea: Lucanidae). Hereditas 133, 249 –253. Halffter, G., Matthews, E.D., 1966. The natural history of dung beetles of the subfamily Scarabaeinae (Coleoptera: Scarabaeidae). Folia Entomolo´gica Mexicana 12–14, 1–312. Hanski, I., Cambefort, Y., 1991. Dung Beetle Ecology, Princeton Univ. Press, Princeton. Hennig, W., 1996. Phylogenetic Systematics, Univ. Illinois Press, Urbana. Howell, W.M., Black, D.A., 1980. Controlled silver staining of nucleolus organizer regions with a protective colloidal developer: a 1-step method. Experientia 36, 1014.
Jekel, H., 1865. Essai sur la classification naturelle des Geotrupes Latreille et descriptions d’espe`ces nouvelles. Ann. Soc. ent. Fr., 4 se´r., v: 513 –618. Lawrence, J.F., Newton, A.F. jr, 1995. Families and subfamilies of Coleoptera (with selected genera, notes, references and data on familygroup names). In: Pakaluk, J., Slipinski, S.A. (Eds.), Biology, Phylogeny, and Classification of Coleoptera: Papers Celebrating the 80th Birthday of Roy A. Crowson, Museum i Instytut Zoologii PAN, Warszawa, pp. 77–106. Levan, A., Fredga, K., Sandberg, A.A., 1964. Nomenclature for centromeric position of chromosomes. Hereditas 52, 201–220. Martı´n-Piera, F., Lo´pez-Colo´n, J.I., 2000. Fauna Iberica, Museo Nacional de Ciencias Naturales, CSIC, Madrid. Moro´n-Rios, M.A., 1984. Escarabajos 200 milliones de an˜os de evolucio´n, nr. 14. Pubblicaciones Instituto de Ecologı´a, Me´xico. Salamanna, G., 1965. Sui cromosomi di Pentodon punctatus Vill Copris hispanus L., e Geotrupes intermedius Costa (Coleoptera, Scarabaeidae), Atti VI Congresso Nazionale Italiano di Entomologia Padova, 11 –14 Settembre 1965, pp. 83 –84. Salamanna, G., 1972. Aspetti della cariologia degli Scarabaeidae (Coleoptera), Atti IX Congresso Nazionale Italiano di Entomologia, p. 313 –326. Schmid, M., 1978. Chromosome banding in Amphibia. II. Constitutive heterochromatin and nucleolus organizer regions in Ranidae, Microhylidae and Rhacophoridae. Chromosoma 68, 131–148. Schmid, M., Haaf, T., Geile, B., Sims, S., 1983. Chromosome banding in Amphibia. VIII. An unusual XY/XX sex chromosome system in Gastrotheca riobambae (Anura, Hylidae). Chromosoma 88, 69 –82. Sumner, A.T., 1972. A simple technique for demonstrating centromeric heterochromatin. Experimental Cell Research 75, 304 –306. Thiriot-Quievreux, C., Insua, A., 1992. Nucleolar organizer region variation in the chromosomes of three oyster species. Journal of Experimental Marine Biology and Ecology 157, 33 –40. Vidal, O.R., Giacomozzi, R.O., 1978. Los cromosomas de la subfamilia Dynastinae (Coleoptera: Scarabaeidae). II. Las bandas en Enema pan (Fabr.). Physis C 38(91), 13–119. Vidal, O.R., Riva, R., Giacomozzi, R.O., 1977. Numeros cromoso´micos de Coleoptera de la Argentina. Physis 37(93), 311 –312. Vitturi, R., Catalano, E., Sparacio, I., Colomba, M., Morello, A., 1996. Multiple sex chromosome systems in the dark beetles Blaps gigas and Blaps gibba (Coleoptera, Tenebrionidae). Genetica 97, 225–233. Vitturi, R., Colomba, M., Barbieri, R., Zunino, M., 1999. Ribosomal location in the scarab beetle Thorectes intermedius (Costa) (Coleoptera: Geotrupidae) using banding and fluorescent in situ hybridization. Chromosome Research 7, 1–6. Vitturi, R., Libertini, A., Armetta, F., Sparacino, L., Colomba, M., 2002. Chromosome analysis and FISH mapping of ribosomal DNA (rDNA), telomeric (TTAGGG)n and (GATA)n repeats in the leech Haemopis sanguisuga (L.) (Annelida: Hirudinea). Genetica 115, 189–194. Yadav, J.S., Pillai, R.K., 1979. Evolution of karyotypes and phylogenetic relationships in Scarabaeidae (Coleoptera). Zoologischer Anzeiger 202, 105 –118. Yadav, J.S., Pillai, R.K., Karamjeet, 1979. Chromosome numbers of Scarabaeidae (Polyphaga: Coleoptera). The Coleopterists Bulletin 33(3), 309 –318. Yadav, J.S., Burra, M.R., Dange, M.P., 1989. Chromosome number and sex-determining mechanism in 32 species of Indian Coleoptera (Insecta). National Academy Science Letters 12, 93–97. Zunino, M., 1984. Sistematica generica dei Geotrupinae (Coleoptera Scarabaeoidea: Geotrupidae), filogenesi della sottofamiglia e considerazioni biogeografiche. Bollettino del Museo Regionale di Scienze Naturali Torino 2(1), 9–162. Zurita, F., Sa´nchez, A., Burgos, M., Jime´nez, R., Diaz De La Guardia, R., 1977. Interchromosomal, intercellular and interindividual variability of NORs studied with silver staining and in situ hybridization method. Experimental Cell Research 167, 227 –240.
Karyotype, banding and rDNA FISH in the scarab beetle Anoplotrupes stercorosus (Coleoptera Scarabaeoidea: Geotrupidae). Description and comparative analysis Mariastella Colombaa,*, Roberto Vitturib, Nicola Volpeb, Antonella Lanninob, Mario Zuninoa a
Istituto di Ecologia e Biologia Ambientale, Universita` di Urbino “Carlo Bo”, via Sasso 75, 61029 Urbino (PU), Italy b Dipartimento di Biologia Animale, Universita` di Palermo, Via Archirafi 18, 90123 Palermo, Italy
Abstract Six specimens of Anoplotrupes stercorosus (Coleoptera Scarabaeoidea: Geotrupidae) were analysed using conventional staining, banding techniques and fluorescent in situ hybridization with a ribosomal probe (rDNA FISH). Detailed karyotype description was also joined to a comparative analysis between present data and those previously reported for Thorectes intermedius [Chromosome Res. 7 (1999) 1]. The two species, both belonging to the tribe Geotrupini, show the same modal number but different autosomal morphology which is in contrast with the high chromosome stability argued for Geotrupinae during the last three decades. Moreover, a detailed comparison reveals the occurrence of a plesiomorphic condition in A. stercorosus with respect to the apomorphic one of T. intermedius. This finding agrees with phylogenetic relationships proposed for the two genera based on morphological and anatomical characters. q 2004 Elsevier Ltd. All rights reserved. Keywords: Coleoptera Scarabaeoidea Geotrupidae; Cytogenetics; 18S rDNA FISH
1. Introduction The coleopteran superfamily Scarabaeoidea, whose origins can be traced back to the late Mesozoic (200 MYA), is a cosmopolitan, monophyletic group which includes about 35,000 described species. Up to the present time, this taxon has been investigated from many different points of view, as demonstrated by the high number of studies (i.e. Halffter and Matthews, 1966; Moro´n-Rios, 1984; Hanski and Cambefort, 1991 and authors quoted therein) reporting on phylogeny, ecology, biogeography, behaviour and reproductive biology of these interesting organisms. On the contrary, other subjects including, among others, immunology, molecular biology and cytogenetics remain mostly unknown. Available karyological data are limited to the haploid and/or diploid numbers of about 200 species, the karyotype description of some of them (Salamanna, 1965, 1972; Vidal et al., 1977; Yadav et al., 1979, 1989) and chromosome banding (silver staining and/or C-banding) of Enema pan (F.) (Dynastinae) (Vidal and Giacomozzi, 1978) and Bubas bison L. and Glyphoderus sterquilinus (Westwood) (Scarabaeinae) (Colomba et al., 1996). Recently, fluorescent * Corresponding author. Tel.: þ 39-722-303-438; fax: þ39-722-303-436. E-mail address: [email protected] (M. Colomba). 0968-4328/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2004.04.004
in situ hybridization with ribosomal sequences as a probe (rDNA FISH) has mapped major ribosomal clusters (18S-28S rDNA) in three species of three different families, Thorectes intermedius (Costa) (Geotrupidae) (Vitturi et al., 1999), Gymnopleurus sturmi McLeay (Scarabaeidae) (Colomba et al., 2000a) and Dorcus parallelipipedus L. (Lucanidae) (Colomba et al., 2000b). The genus Anoplotrupes (Geotrupidae) was previously described by Jekel (1865) as a subgenus of Geotrupes. According to Zunino (1984), within the tribe Geotrupini, this genus shows a close phylogenetic relationship with the Asiatic genera Glyptogeotrupes Nikolaev and Epigeotrupes Bovo and Zunino. It comprises few species distributed in North America (North of Mexico), with the exception of A. stercorosus (Scriba, 1790) whose distribution covers the European fraction of the Palearctic Region with a frequency gradient decreasing southwards (Zunino, 1984; Martı´n-Piera and Lopez-Colo´n, 2000). In Mediterranean areas, it mainly occurs in semi deciduous or pine woods (at high altitudes). In the present study, we provide an extensive cytogenetic analysis of A. stercorosus using both classical (silver staining, C-banding and fluorochrome staining) and molecular (rDNA FISH) methods. Results are also related with the corresponding ones of another Geotrupini, T. intermedius, previously investigated by Vitturi et al. (1999).
718
M. Colomba et al. / Micron 35 (2004) 717–720
Such a comparison represents an initial contribution to the reconstruction of chromosomal changes that have occurred during the Geotrupidae evolution.
2. Materials and methods Six Anoplotrupes stercorosus (four males and two females), identified according to Zunino (1984); Baraud (1992), were collected at the end of May, 2001 in the beech wood near Busana (Reggio Emilia, North Italy, in the ‘Parco Regionale dell’Alto Appennino Reggiano’). Chromosome preparations were obtained by the airdrying technique after in vivo colchicine (0.15%) treatment. Unfortunately, females did not provide good-quality material for chromosome analysis, therefore, data reported herein resulted from the males. Slides were subjected to Giemsa staining (Vitturi et al., 1996), C-banding (Sumner, 1972), silver staining (Ag-NOR) (Howell and Black, 1980), DAPI and CMA3 staining (Schmid et al., 1983) and rDNA FISH (Colomba et al., 2000b). Chromosomes were classified on the basis of the criteria proposed by Levan et al. (1964). rDNA FISH was carried out on fixed spermatocyte chromosomes using the 18S rDNA of the sea urchin Paracentrotus lividus (Echinodermata) as a probe. Nick translation labelling of the probe with digoxigenin and hybridization experiments were performed according to manufacturer’s (Roche) instructions. Slides were mounted in an antifade solution containing propidium iodide (PI) (5 mg/ml) and viewed under a Leica I3 filter set (BP 450490; LP 515). Microphotographs were taken using a Leica microscope on Kodak Ektacolor 800 ASA film. The systematic scheme adopted in the present paper is that proposed by Lawrence and Newton (1995) for Scarabaeoidea, and that of Zunino (1984) for Geotrupidae.
ranged from about 4.8 to 1.8 mm, showed meta-/submetacentric (M/SM) (pairs no. 3, 4, 5, 6, 8) and subtelo-/acrocentric (ST/A) (pairs no. 1, 2, 7, 9, 10) morphologies. The sex pair consisted of two small elements (M and ST), about 1.4 mm long, whose identification was impossible, since, due to the lack of female chromosome preparations, we have not been able to conclusively distinguish the X from the Y. C-banding showed that heterochromatin coincided with one or both telomeric regions in nearly all chromosomes (18 spreads analysed) (Fig. 2). In a few chromosomes a small heterochromatic dot was located at the centromeric region (Fig. 2, see arrow). Silver staining pattern was repetitive and consisted of argentophilic dots corresponding to heterochromatic regions (28 spermatogonial metaphases analysed) (Fig. 3). A diffuse silver stainability also characterized bivalents at early (Fig. 4A) and late (Fig. 4B) metaphase I stage. Both in mitotic (Fig. 3, see arrows) and meiotic (Fig. 4B, see arrow) spreads, the elements of the sex pair were distinguished by their large silver deposits. CMA3 staining (GC-specific) evidenced two mediumlarge elements—probably the NOR bearing ones—with a bright paracentromeric area (Fig. 5A), whereas DAPI (ATspecific) stained all chromosomes homogeneously (Fig. 5B). rDNA FISH constantly mapped major ribosomal sites in correspondence of the CMA3 positive regions of the two medium-large chromosomes (25 spreads analyzed). Observed hybridized areas significantly varied in dimension, resulting either both large (cytotype A) (10 spreads) (Fig. 6A), or different in size (cytotype B) (15 spreads) (Fig. 6B), thus implying the occurrence of an intercellular variability in the number of ribosomal loci. This finding will not be discussed further since polymorphism in the copy number of rDNA genes is well-known for many animal taxa (Zurita et al., 1977).
4. Discussion 3. Results Giemsa staining of spermatogonial metaphases (32 spreads) of A. stercorosus gave the diploid chromosome number ð2nÞ of 22 which confirms that previously reported by Yadav et al. (1979) for a different strain of the same species (sub Geotrupes). Chromosomes, paired according to their length and centromere position, were arranged into 11 pairs, including ten autosomes and the heteromorphic sex elements (XY) (10 spreads analysed) (Fig. 1). Autosomes whose dimensions
In the last thirty years it has been maintained that karyotypic homogeneity occurs within Geotrupinae (Salamanna, 1972; Yadav and Pillai, 1979). Taking into account that available data on scarab beetles were obtained by dated and non-specific analyses, the present study was carried out to test the reliability of this hypothesis. The diploid number ð2n ¼ 22Þ and the sex-determining mechanism (XX/XY) of A. stercorosus are consistent with those reported for eight Geotrupinae species studied so far (Yadav et al., 1979; Colomba, 1998; Vitturi et al., 1999).
Fig. 1. Karyotype of Anoplotrupes stercorosus obtained from a Giemsa stained spermatogonial metaphase (2n ¼ 22; 10AA þ XY; 11 M/SM þ 11 ST/A). The Fondamental number (FN), on the basis of chromosome morphology described in the results section, is FN ¼ 33 (M/SM chromosomes and ST/A chromosomes are considered as biarmed and monoarmed, respectively).
M. Colomba et al. / Micron 35 (2004) 717–720
Fig. 2. C-banded mitotic chromosomes of A. stercorosus.
Such a homogeneity, evidenced by a low resolution investigation, steadily decreases when a more accurate analysis is performed. In fact, within the tribe Geotrupini, a comparison between A. stercorosus and T. intermedius karyotypes reveals marked differences in chromosome morphology. Apart from the sex elements which show very similar morphology and dimension in both species, autosomes consist of five biarmed and five monoarmed pairs in A. stercorosus, and of 10 monoarmed pairs in T. intermedius. This finding refutes the high stability in chromosome morphology previously argued for the subfamily Geotrupinae (Salamanna, 1972). A lack of a karyological homogeneity within Geotrupinae is largely confirmed when analysis is extended to: 1. NOR number. Major ribosomal clusters are mapped by FISH on one chromosome pair in A. stercorosus and on two chromosome pairs in T. intermedius (Vitturi et al., 1999); 2. heterochromatin location. After C-banding, heterochromatin is centromeric in T. intermedius (Vitturi et al., 1999), whereas it is mostly telomeric in A. stercorosus; 3. heterochromatin base-pair composition. After CMA3 staining, all heterochromatin including both constitutiveand NOR-associated one, is highly compartmentalized in GC base pairs in T. intermedius (Vitturi et al., 1999) whereas, in A. stercorosus, GC-rich base pairs correspond only to NOR-associated heterochromatin. This finding extends the data in previous papers (Colomba et al., 1996, 2000a,b; Vitturi et al., 1999) in which it has been shown that advanced cytogenetic techniques can reveal a high karyological diversification within Scarabaeoidea.
Fig. 3. Silver stained spermatogonial chromosomes of A. stercorosus (arrows indicate the sex chromosomes).
719
Fig. 4. Silver stained meiotic chromosomes of A. stercorosus. Early metaphase I (A); and late metaphase I (B) (arrow indicates the sex bivalent showing large silver deposits).
As already demonstrated for all scarab beetles species so far investigated, in A. stercorosus the silver stainability of all chromosomes was the result of an extensive Ag-positive reaction of heterochromatin. Such non-specific staining prevented unequivocal identification of NORs, so major ribosomal sites were mapped by rDNA FISH. CMA3 staining yielded more information than silver impregnation since, in line with rDNA FISH pattern, it showed NORs on one chromosome pair. The discrepancy between silver and chromomycin patterns is the result of a varying reaction of heterochromatic DNA (entirely silverpositive) to the fluorochrome. In fact, only the NORassociated heterochromatin fluoresced brightly, while the constitutive one reacted homogeneously. This heterogeneous response to CMA3 staining in A. stercorosus disagrees with results obtained in all Scarabaeoidea species analyzed so far, in which both types of heterochromatic DNA react in the same way. In particular, heterochromatin was found to be CMA3-positive in eight species (Colomba, 1998, 2000a; Vitturi et al., 1999), CMA3-negative in 1 species (Colomba, 1998), CMA3-neutral in two species (Colomba, 1998; Colomba et al., 2000b) and DAPI-positive in one species (Colomba et al., 1996). Finally, it is maintained that the occurrence of two NORs and a low level or the absence of chromatin compartmentalization are to be considered as the basic karyotypic form in most vertebrates (Schmid, 1978) and some invertebrates (Thiriot-Quievreux and Insua, 1992; Vitturi et al., 2002). If we extend this assumption to the tribe Geotrupini, A. stercorosus shows primitive karyotipic characters
Fig. 5. Fluorochrome stained spermatogonial metaphase plates of A. stercorosus. (A) CMA3 evidenced a bright paracentromeric area in each element of one chromosomes pair; (B) DAPI stained homogeneously all chromosomes.
720
M. Colomba et al. / Micron 35 (2004) 717–720
Fig. 6. rDNA FISH of A. stercorosus spermatogonial chromosomes. The micrographs were taken with a Leica I3 filter set allowing the simultaneous visualization of the PI-stained chromosomes (red colour) and the hybridization sites of the 18S rDNA probe (yellow colour) Hybridization signals are similar in size (A); and different in size (B) (scale bar: 16 mm ¼ 10 mm).
(two NORs and uncompartmentalized heterochromatin) when compared to T. intermedius (more than two NORs and evident GC-rich heterochromatin). This conclusion agrees with the phylogeny of Geotrupini proposed by Zunino (1984). In this scheme, based on numerous morphological and anatomical characters analyzed according to criteria proposed by Hennig (1996), six genera among which is included Anoplotrupes, represent the sister-group, relatively primitive, of the phyletic branch leading to the genera Thorectes and Trypocopris. Acknowledgements This work was supported by a MURST fund (Fondi di Ricerca Scientifica ex 60%) to M. Colomba. References Baraud, J., 1992. Cole´opte`res Scarabaeoidea d’ Europe, Fe´de´ration Franc¸aise des Sciences Naturelles et Socie´te´ Linne´enne, Lyon. Colomba, M., 1998. L’organizzazione del cariotipo nei Coleotteri Scarabaeoidea: materiali per un’analisi evolutiva. PhD Dissertation. Dipartimento di Biologia Animale. Universita` di Palermo, Palermo, Italy. Colomba, M., Monteresino, E., Vitturi, R., Zunino, M., 1996. Characterization of mitotic chromosomes of the scarab beetles Glyphoderus sterquilinus (Westwood) and Bubas bison (L.) (Coleoptera: Scarabaeidae) using conventional and banding techniques. Biologisches Zentralblatt 115, 58 –70. Colomba, M., Vitturi, R., Zunino, M., 2000a. Karyotype analysis, banding and fluorescent in situ hybridization in the scarab beetle Gymnopleurus sturmi McLeay (Coleoptera Scarabaeoidea: Scarabaeidae). The Journal of Heredity 91(3), 260 –264. Colomba, M., Vitturi, R., Zunino, M., 2000b. Chromosome analysis and rDNA FISH in the stag beetle Dorcus parallelipipedus L. (Coleoptera: Scarabaeoidea: Lucanidae). Hereditas 133, 249 –253. Halffter, G., Matthews, E.D., 1966. The natural history of dung beetles of the subfamily Scarabaeinae (Coleoptera: Scarabaeidae). Folia Entomolo´gica Mexicana 12–14, 1–312. Hanski, I., Cambefort, Y., 1991. Dung Beetle Ecology, Princeton Univ. Press, Princeton. Hennig, W., 1996. Phylogenetic Systematics, Univ. Illinois Press, Urbana. Howell, W.M., Black, D.A., 1980. Controlled silver staining of nucleolus organizer regions with a protective colloidal developer: a 1-step method. Experientia 36, 1014.
Jekel, H., 1865. Essai sur la classification naturelle des Geotrupes Latreille et descriptions d’espe`ces nouvelles. Ann. Soc. ent. Fr., 4 se´r., v: 513 –618. Lawrence, J.F., Newton, A.F. jr, 1995. Families and subfamilies of Coleoptera (with selected genera, notes, references and data on familygroup names). In: Pakaluk, J., Slipinski, S.A. (Eds.), Biology, Phylogeny, and Classification of Coleoptera: Papers Celebrating the 80th Birthday of Roy A. Crowson, Museum i Instytut Zoologii PAN, Warszawa, pp. 77–106. Levan, A., Fredga, K., Sandberg, A.A., 1964. Nomenclature for centromeric position of chromosomes. Hereditas 52, 201–220. Martı´n-Piera, F., Lo´pez-Colo´n, J.I., 2000. Fauna Iberica, Museo Nacional de Ciencias Naturales, CSIC, Madrid. Moro´n-Rios, M.A., 1984. Escarabajos 200 milliones de an˜os de evolucio´n, nr. 14. Pubblicaciones Instituto de Ecologı´a, Me´xico. Salamanna, G., 1965. Sui cromosomi di Pentodon punctatus Vill Copris hispanus L., e Geotrupes intermedius Costa (Coleoptera, Scarabaeidae), Atti VI Congresso Nazionale Italiano di Entomologia Padova, 11 –14 Settembre 1965, pp. 83 –84. Salamanna, G., 1972. Aspetti della cariologia degli Scarabaeidae (Coleoptera), Atti IX Congresso Nazionale Italiano di Entomologia, p. 313 –326. Schmid, M., 1978. Chromosome banding in Amphibia. II. Constitutive heterochromatin and nucleolus organizer regions in Ranidae, Microhylidae and Rhacophoridae. Chromosoma 68, 131–148. Schmid, M., Haaf, T., Geile, B., Sims, S., 1983. Chromosome banding in Amphibia. VIII. An unusual XY/XX sex chromosome system in Gastrotheca riobambae (Anura, Hylidae). Chromosoma 88, 69 –82. Sumner, A.T., 1972. A simple technique for demonstrating centromeric heterochromatin. Experimental Cell Research 75, 304 –306. Thiriot-Quievreux, C., Insua, A., 1992. Nucleolar organizer region variation in the chromosomes of three oyster species. Journal of Experimental Marine Biology and Ecology 157, 33 –40. Vidal, O.R., Giacomozzi, R.O., 1978. Los cromosomas de la subfamilia Dynastinae (Coleoptera: Scarabaeidae). II. Las bandas en Enema pan (Fabr.). Physis C 38(91), 13–119. Vidal, O.R., Riva, R., Giacomozzi, R.O., 1977. Numeros cromoso´micos de Coleoptera de la Argentina. Physis 37(93), 311 –312. Vitturi, R., Catalano, E., Sparacio, I., Colomba, M., Morello, A., 1996. Multiple sex chromosome systems in the dark beetles Blaps gigas and Blaps gibba (Coleoptera, Tenebrionidae). Genetica 97, 225–233. Vitturi, R., Colomba, M., Barbieri, R., Zunino, M., 1999. Ribosomal location in the scarab beetle Thorectes intermedius (Costa) (Coleoptera: Geotrupidae) using banding and fluorescent in situ hybridization. Chromosome Research 7, 1–6. Vitturi, R., Libertini, A., Armetta, F., Sparacino, L., Colomba, M., 2002. Chromosome analysis and FISH mapping of ribosomal DNA (rDNA), telomeric (TTAGGG)n and (GATA)n repeats in the leech Haemopis sanguisuga (L.) (Annelida: Hirudinea). Genetica 115, 189–194. Yadav, J.S., Pillai, R.K., 1979. Evolution of karyotypes and phylogenetic relationships in Scarabaeidae (Coleoptera). Zoologischer Anzeiger 202, 105 –118. Yadav, J.S., Pillai, R.K., Karamjeet, 1979. Chromosome numbers of Scarabaeidae (Polyphaga: Coleoptera). The Coleopterists Bulletin 33(3), 309 –318. Yadav, J.S., Burra, M.R., Dange, M.P., 1989. Chromosome number and sex-determining mechanism in 32 species of Indian Coleoptera (Insecta). National Academy Science Letters 12, 93–97. Zunino, M., 1984. Sistematica generica dei Geotrupinae (Coleoptera Scarabaeoidea: Geotrupidae), filogenesi della sottofamiglia e considerazioni biogeografiche. Bollettino del Museo Regionale di Scienze Naturali Torino 2(1), 9–162. Zurita, F., Sa´nchez, A., Burgos, M., Jime´nez, R., Diaz De La Guardia, R., 1977. Interchromosomal, intercellular and interindividual variability of NORs studied with silver staining and in situ hybridization method. Experimental Cell Research 167, 227 –240.