Cytogenetic analysis of Astylus antis (Perty, 1830) (Coleoptera, Melyridae): Karyotype, heterochromatin and location of ribosomal genes

Cytogenetic analysis of Astylus antis using mitotic and meiotic cells was performed to characterize the haploid and diploid numbers, sex determination system, chromosome morphology, constitutive heterochromatin distribution pattern and chromosomes carrying nucleolus organizer regions (NORs). Analysis of spermatogonial metaphase cells revealed the diploid number 2n = 18, with mostly metacentric chromosomes. Metaphase I cells exhibited 2n = 8II+Xyp and a parachute configuration of the sex chromosomes. Spermatogonial metaphase cells submitted to C-banding showed the presence of small dots of constitutive heterochromatin in the centromeric regions of nearly all the autosomes and on the short arm of the X chromosome (Xp), as well as an additional band on one of the arms of pair 1. Mitotic cells submitted to double staining with base-specific fluorochromes (DAPI-CMA3 ) revealed no regions rich in A+T or G+C sequences. Analysis of spermatogonial mitotic cells after sequential Giemsa/AgNO 3 staining did not reveal any specific mark on the chromosomes. Meiotic metaphase I cells stained with silver nitrate revealed a strong impregnation associated to the sex chromosomes, and in situ hybridization with an 18S rDNA probe showed ribosomal cistrons in an autosomal bivalent.


Introduction
The suborder Polyphaga is the most numerous of the order Coleoptera and displays the greatest structural and biological diversity of species (Gillot, 1995;Costa, 1999). The superfamily Cleroidea belongs to the suborder Polyphaga and comprises approximately 10,000 taxonomically described species (Costa, 2003), distributed among eight families (Lawrence and Newton, 1995), such as Melyridae, with approximately 5000 species, 68 of which occur in the Neotropical region (Costa, 2003). The genus Astylus belongs to the family Melyridae and includes a number of Brazilian species, such as Astylus antis, A. quadrilineatus, A. sexmaculatus and A. variegatus, which are best known for visiting the flowers of cultivated plants, such as corn, cotton and sorghum (Rosseto and Rosseto, 1976;Souza and Carvalho, 1994;Ventura et al., 2007).
In the superfamily Cleroidea, only 16 species have been cytogenetically analyzed, 12 species belonging to the family Cleridae and four species of the family Melyridae. The 12 species of Cleridae, distributed among five genera (Enoclerus, Priocera, Thanasimus, Trichodes and Necrobia), exhibit karyotype uniformity, i.e., 2n = 18, the basal sex determination system for Coleoptera, Xyp, and meta/submetacentric morphology for all chromosomes (Smith, 1953;Virkki, 1963;Smith and Virkki, 1978;Yadav and Dange, 1989;Schneider et al., 2007a). However, the four species belonging to the family Melyridae display differences both in their chromosome number and their sex determination system (Smith and Virkki, 1978). Chromosome morphology was only described for A. variegates, in which all chromosomes are metacentric (Schneider et al., 2007a).
Cytogenetic data using differential staining in species of Cleroidea are limited to A. variegatus, which has small blocks of heterochromatin in the pericentromeric region of all chromosomes, except Xyp. In this species, the nucleolus organizer region (NOR) is located in autosome pair 2 (Schneider et al., 2007a).
In Coleoptera with the Xyp sex determination system, it is common to find nucleolus material associated to the sex chromosomes. Furthermore, there are a number of different mechanisms described to explain the association and segregation of these chromosomes in meiosis, depending on their degree of differentiation. These mechanisms oscillate between a nucleolus association and/or a synaptic association (Smith and Virkki, 1978;Juan et al., 1993;Petitpierre, 1996). In Coleoptera, however, NORs are located in either autosomes and/or sex chromosomes (Almeida et al., 2000;Schneider et al., 2007aSchneider et al., , 2007b. The few studies that have employed fluorescent in situ hybridization (FISH) in Coleoptera have found conflicting results between the location of the rDNA genes and the silver staining, particularly regarding the sex chromosomes of the Xyp system in some species. Therefore, the nucleolus theory for the maintenance and segregation of the sex chromosomes belonging to this system (Weber, 1971;Drets et al., 1983;Virkki, 1983;Postiglioni and Brum-Zorilla, 1988;Postiglioni et al., 1991;Juan et al., 1993;Maffei et al., 2001) has been questioned Moura et al., 2003;Schneider et al., 2007aSchneider et al., , 2007b.
In order to understand how chromosome evolution occurred in the different species of this group, it is important to establish the constitutive heterochromatin distribution pattern and to identify the NOR-bearing chromosomes. These were the aims of the present study, in addition to chromosomally characterize the species Astylus antis, using both the mitotic karyotype and meiotic cells.
Cytological preparations were obtained from the gonads of adult male individuals. The gonads were removed in insect saline solution, treated with hypotonic solution (tap water) for six minutes and fixed in Carnoy I. Then, the gonads were macerated in 45% acetic acid solution, and the slides were dried on a metal plate at a temperature of 35 to 40°C; later on, the slides were stained with 3% Giemsa in pH 6.8 phosphate buffer for 15 min.
The C-banding and base-specific fluorochrome staining (DAPI/CMA 3 ) methods described by Sumner (1972) and Schweizer (1980), respectively, were used to determine the distribution and the AT/GC content of the constitutive heterochromatin. The silver nitrate impregnation method described by Howell and Black (1980) and the fluorescent in situ hybridization (FISH) method with 18S rDNA described by Pinkel et al. (1986) were used to identify the chromosomes bearing NORs. The partial 18S rDNA probe (732 pb) was obtained through amplification by PCR labeled with biotin-14-dATP hapten (Invitrogen), using the cloned 18S fragment of Omophoita octoguttata (Coleoptera) as template. The hybridization signals were detected using avidin-fluorescein isothiocyanate (Avidin-FITC, Sigma). For amplification of the signals, we used anti-avidin biotinylated (Sigma) and Avidin-FITC (Sigma) conjugated antibodies. Overall hybridization was performed under high stringency conditions (2.5 ng/mL probes, 50% deionized formamide, 10% dextran sulfate, 2XSSC at 37°C overnight). After hybridization, the slides were washed in 15% formamide/0.2XSSC at 42°C for 20 min, 0.1XSSC at 60°C for 15 min, and 4XSSC /0.05% Tween at room temperature for 10 min, the latter consisting of two washes of 5 min each. Chromosomes were counterstained with DAPI (0.2 mg/mL) in anti-fade solution.
Approximately 40 cells from each specimen were examined. Chromosomes were counted and identified whenever possible. The best mitotic and meiotic cells in both conventional and differential staining were photographed under an optical photomicroscope (Olympus BX41), with a 100x immersion objective. The metaphase cells submitted to the base-specific fluorochromes and FISH were photographed with a digital camera (Olympus C-5060 5.1 Megapixel) with specific filters, or recorded by real-time digital imaging with a DP-71 camera and DP controller software.
The karyotypes were arranged and numbered in decreasing order, based on size and morphology of the chromosomes, and the homologous chromosomes were tentatively paired to facilitate presentation and comparison, as proposed by Levan et al. (1964).

Conventional staining
Analysis of spermatogonial metaphase cells revealed the diploid chromosome complement 2n = 18 = 16+Xyp. Most of the autosomes were metacentric, only pairs 5 and 7 were submetacentric. The Xp chromosome was submetacentric, and the yp chromosome was extremely small, which made it impossible to determine its morphology but it may be acrocentric (Figure 1a).
The pachytene cells showed all bivalents completely, including the sex chromosomes, which displayed a parachute configuration. Small positive heteropycnotic blocks were found in these cells (Figure 2a). The study of diplotene cells revealed the occurrence of one or two chiasmata per bivalent (Figure 2b). The metaphase I cells examined showed the chromosome meioformula 2n = 8II+Xyp and the parachute configuration of the sex chromosomes (Figure 2c). The metaphase I cells showed a haploid complement n = 8+Xp or n = 8+yp (Figures 2d and 2e), indicating normal chromosome segregation during anaphase I. The yp chromosome exhibited negative heteropycnosis in the majority of meiotic phases analyzed.

Differential staining
C-banding and base-specific fluorochrome (DAPI/CMA 3 ) staining of spermatogonial metaphase cells revealed the presence of small dots of heterochromatin in the centromeric regions of most autosomes, as well as an interstitial band on one of the arms of the pair 1 chromosomes and on the short arm of the Xp chromosome (Figure 1b), but with no differentiation between AT-and GC-rich sites (data not shown). Sequential Giemsa/AgNO 3 staining of spermatogonial mitotic cells revealed no NOR-specific labeling on the chromosomes.
Meiotic cells submitted to C-banding and basespecific fluorochrome staining showed no specific AT-or GC-rich blocks or sites. Sequential Giemsa/AgNO 3 staining of meiotic metaphase I cells revealed strong silver nitrate impregnation on the sex chromosomes (Figures 3a and  3b). This block was interpreted as argyrophilic material. FISH analysis of pachytene and metaphase I cells using an 18S rDNA probe revealed a fluorescent signal strongly associated to an autosomal bivalent and no labeling on the sex chromosomes (Figures 3c and 3d).

Discussion
The results obtained for Astylus antis regarding the sex determination system, chromosome morphology, C-band distribution pattern and location of NORs are in agreement with those described for many other species of Coleoptera. The 2n = 18 = 16+Xyp chromosome number is similar to that described for the 12 species of Cleridae (Smith, 1953;Virkki, 1963;Smith and Virkki, 1978;Yadav and Dange, 1989), whereas it is in agreement with only one of the four Melyridae species analyzed -A. variegatus (Schneider et al., 2007a). Furthermore, the chromosome Cytogenetic analysis of Astylus antis 239  formula 2n = 18 differs from the 2n = 20 = 18+Xyp described as basal for the order by Smith (1950) and supported by a number of recent studies (Maffei et al., 2000;2001;Moura et al., 2003;Rozek et al., 2004;Almeida et al., 2000;Schneider et al., 2007aSchneider et al., , 2007b. The difference between the chromosome number found and the basal number may be explained by the occurrence of fusion-type chromosome rearrangements between two pairs of autosomes, followed by pericentric inversion, as also proposed for A. variegatus by Schneider et al. (2007a).
According to Smith and Virkki (1978), the evolutionary tendency for Coleoptera of the suborder Polyphaga was to maintain the chromosome number close to the original, whereas, for representatives of Adephaga, the tendency was to increase the chromosome number through autosomal centric fission. However, centric fusion events appear to be less frequent, as there are only few species with low chromosome numbers.
In the few cytogenetically studied species from the family Cleridae, no change in sex chromosomes was observed, so the Xyp sex determination system was maintained. Nevertheless, in the family Melyridae, maintenance of the Xyp system has been observed in Hoppigiana hudsonica (2n = 6II+Xyp) and A. variegatus (2n = 8II+Xyp), while Collops sp (2n = 8II+X0) and Endeodes collaris (2n = 9II+X0) lost the yp chromosome, giving rise to an X0 sex determination system (Smith, 1953;Virkki, 1963;Smith and Virkki, 1978;Yadav and Dange, 1989;Schneider et al., 2007a). Small karyotype differences were found when comparing the results from A. antis with the description for A. variegatus regarding the metacentric morphology of all the chromosomes, the behavior of the sex chromosomes and the presence of B chromosomes (Schneider et al., 2007a). Thus, it can be inferred that small rearrangements of the inversion type and differentiation of the sex chromosomes occurred during the chromosome differentiation of these species.
Negative heteropycnosis as observed in the yp chromosome of A. antis has also been found in some species of Coleoptera, including A. variegatus. Differences involving heteropycnosis may occur due to differential chromosome condensation and/or the presence of a special type of chromatin (Virkki, 1967;Yadav et al., 1985;Almeida et al., 2000). The number of chiasmata found in A. antis is in agreement with the number described for most species of Coleoptera, as well as for Enoclerus sp, Necrobia ruficollis and Astylus variegatus, which belong to the superfamily Cleroidea (Virkki, 1963;Yadav and Dange, 1989;Schneider et al., 2007a).
From the results obtained with the double staining (DAPI/CMA 3 ), which coincide with those obtained by Cbanding, it can be concluded that the weakly fluorescent signals are heterochromatic regions, but with no differentiation between AT-and GC-rich sites. A number of studies have attempted to explain the conflicting results between the content of DNA bases and responses to base-specific fluorochromes (Comings and Drets, 1976;Saitoh and Laemmli, 1994;Vicari et al., 2008). The fluorochrome DAPI binds to DNA, but its fluorescence is significantly enhanced in AT-rich domains. According to Comings and Drets (1976), Comings (1978) and Johnston et al. (1978), the antibiotic daunomycin only emits fluorescence when the AT content exceeds 65%. According to Vicari et al. (2008), the absence of fluorescence on large heterochromatic blocks of the fish Astyanax janeiroensis is due to the 240 Mendes-Neto et al. effect of competition between two families of repetitive DNA co-located in the same chromosome domains. Contrarily, competition and/or excitation energy transference between DAPI and CMA 3 , together with the absence of AT/GC differentiation in these regions (Zimmer et al., 1971) and the state of heterochromatic compaction, could explain the coinciding results of the C-bands and the DAPI-CMA 3 fluorochrome signals (Saitoh and Laemmli, 1994). Moura et al. (2003) obtained similar results using triple CMA 3 /DA/DAPI staining: they found that in Phyllophaga (Phytalus) vestita there was no difference between the CMA 3 and DAPI signals, both of which were positive. Lyogenys fuscus displays a strong fluorescent signal by DAPI in the pericentromeric region of all chromosomes. Likewise, Vitturi et al. (1999) found that positive CMA 3 regions coinciding with C bands were also DAPIpositive in Thorectes intermedius (Geotrupidae).
Analyzing Epilachna paenulata with the fluorochrome method (Quinacrine HCl or Hoechst 33258), Drets et al. (1983) found intensely fluorescent regions in the centromeric region of the autosomes, a region rich in AT sequences. Juan et al. (1991) studied Tenebrio molitor and Plohl et al. (1993) analyzed testicular cells from Tribolium confusum using DA/DAPI fluorochromes and found that the pericentromeric and centromeric regions of all chromosomes in the complement were rich in AT sequences.
FISH with the 18S rDNA probe revealed a fluorescence signal strongly associated to an autosomal bivalent in Astylus antis. The labeling obtained by silver nitrate staining on the sex chromosomes without the presence of ribosomal cistrons (which were detected by FISH only in one autosomal bivalent) in A. antis is in agreement with the results obtained for other species of Coleoptera with the Xyp system (Vitturi et al., 1999;Colomba et al., 2000a;Moura et al., 2003;Bione et al., 2005). It is also in agreement with a survey carried out by Schneider et al. (2007b), in which 81% of Adephaga and Polyphaga species had NORs located on the autosome pairs. The silver staining of these nonspecific blocks may result from the presence of an argyrophilic substance, which theoretically facilitates the configuration, maintenance and segregation of the sex chromosomes of the Xyp system, as described by a number of authors (Virkki et al., 1990(Virkki et al., , 1991Juan et al., 1993;Petitpierre, 1996;Moura et al., 2003;Bione et al., 2005;Schneider et al., 2007aSchneider et al., , 2007b. A large number of studies have associated these nonspecific silver nitrate marks with argyrophilic proteins and heterochromatic regions, particularly proteins associated to these regions (Virkki et al., 1991;Vitturi et al., 1999;Colomba et al., 2000aColomba et al., , 2000bColomba et al., , 2004Colomba et al., , 2006Bione et al., 2005).
The karyotype differences observed regarding chromosome morphology, C-banding patterns and behavior of the sex chromosomes in meiosis of A. antis in comparison to the description of A. variegatus suggest that the karyotype evolution of these two species may have involved different types of chromosome rearrangements, such as small inversions and the addition of heterochromatin. Regarding the plesiomorphic characteristics for the order, the reduction in number may have occurred due to pericentric inversion, followed by fusion between autosomes, with no involvement of the sex chromosomes.