Karyotype characterization, constitutive heterochromatin and nucleolus organizer regions of Paranaita opima (Coleoptera, Chrysomelidae, Alticinae) Mara Cristina de Almeida1, Carlos Campaner2 and Doralice Maria Cella3 1Departamento de Biologia Estrutural, Molecular e Genética, Setor de Ciências Biológicas e da Saúde, Universidade Estadual de Ponta Grossa, Ponta Grossa, PR, Brazil. 2Museu de Zoologia da Universidade de São Paulo, São Paulo, SP, Brazil. 3Universidade Estadual Paulista, UNESP, Instituto de Biociências, Departamento de Biologia, Rio Claro, SP, Brazil. Abstract Species of the subtribe Oedionychina not only have a highly uniform diploid number of 2n = 22 (20+X+y) but have the karyotypic peculiarity of possessing extremely large sex chromosomes. We analyzed Paranaita opima embryos and gonadal cells to determine their diploid number, chromosomal morphology, type of sex determination system, consti- tutive heterochromatin pattern and which chromosomes bear nucleolus organizer regions (NORs). The diploid num- ber of P. opima was 2n = 22 (20+XY/XX) with all chromosomes being metacentric. Chromosome pair 6 showed an interstitial secondary constriction on the short arm. The C-banding technique revealed centromeric constitutive heterochromatin in all chromosomes, which, in pair 6, extended up to the secondary constriction of the short arm, ad- ditional C-bands also being present on the Y chromosome. Silver nitrate nucleolar organizer region (Ag-NOR) stain- ing showed NORs on the secondary constriction of pair 6. Fluorochrome analysis with chromomycin A3 (CMA3), 4’-6-diamidino-2-phenylindole (DAPI) and the distamycin A (DA) counterstain showed that the short arm of chromo- some pair 6 exhibited a GC-rich block extending from the proximal to the median region, including part of the second- ary constriction. The same techniques also showed AT-rich blocks at the centromeric region of all chromosomes and at the terminal region of the short arm of pair 6. The basic karyotype characteristics and C band pattern of P. opima are similar to those described for other species in the subtribe Oedionychina. The pattern of autosomal NORs ob- served in P. opima corresponds to that registered in the majority of the Chrysomelidae species. Key words: C bands, chromosome, embryos, fluorochromes, NOR. Received: July 4, 2005; Accepted: December 12, 2005. Introduction The tribe Oedionychini (Alticinae) includes two sub- tribes, Disonychina and Oedionychina. Cytogenetic studies performed on 20 Disonychina species have shown that this group possesses high inter- and intraspecific variability in relation to the diploid number and type of sex determina- tion system. An idea of the extent of such variation is given by the karyotypic variability described in the Disonycha species D. spilotracheta (2n = 29 (13II+X1y+X2), Virkki, 1988b), D. bicarinata (2n = 64 (30II+X1Y+X2X3) Virkki, 1988b), D. nigrita (2n = 34 (16II+X+x) Virkki, 1964 and 2n = 33 (15II+X1y+x2) Virkki, 1988b) as well as Phenrica austriaca which has been variously described as having a karyotype of 2n = 49 (22II+Xy+3y) by Virkki (1970) and 2n = 49 (22II+X1y+X2+X3+X4) by Virkki (1988b). In this group, the chromosomal morphology is predominantly metacentric and the sex determination system is variable, with frequent occurrence of multiple systems, involving mainly the X chromosome (Smith and Virkki, 1978; Vidal, 1984; Virkki, 1988b). In contrast, 74 of the 113 species of the Oedionychina subtribe which have been cytogenetically analyzed have shown high karyotype uniformity concerning the diploid number and type of sex determination system, which is 2n = 22 (20+X+y) with the sex chromosomes being asynap- tic during meiosis (Smith and Virkki, 1978; Virkki, 1970, 1971, 1988a, 1989). In the Oedionychina, diploid numbers higher than 2n = 22 are due to an increase in the number of y chromosome, while numbers lower than 2n = 22 are due to alterations involving autosomes. In spite of the great num- ber of Oedionychina species studied, little information Genetics and Molecular Biology, 29, 3, 475-481 (2006) Copyright by the Brazilian Society of Genetics. Printed in Brazil www.sbg.org.br Send correspondence to Mara Cristina de Almeida. Departamento de Biologia Estrutural, Molecular e Genética, Setor de Ciências Biológicas e da Saúde, Universidade Estadual de Ponta Grossa, Av. Carlos Cavalcanti 4748, Uvaranas, 84.030-900 Ponta Grossa, PR, Brazil. E-mail: almeidamara@uol.com.br. Research Article exists on their chromosomal morphology, making it impos- sible to establish a morphological pattern for the subtribe. Of the 18 Oedionychina species whose chromosomes have been morphologically classified, about 50% have exhibited solely metacentric autosomes, 25% only acrocentric auto- somes and the other 25% metacentric and acrocentric auto- somes with a predominance of metacentrics (Smith and Virkki, 1978; Virkki, 1985, 1989; Virkki et al. 1991; Virkki and Santiago-Blay, 1993, 1996). In all these species, the X chromosome is invariably metacentric and the y chromo- some is predominantly metacentric, with the exception of two species in which the y chromosome is acrocentric. A peculiarity of all Oedionychini species is the pres- ence of extremely large sex chromosomes, sometimes cor- responding to 50% of the entire genome (Virkki, 1985). In general, the chromosomes of the Oedionychini species have been analyzed by means of standard staining, and only 7 species have been studied with regard to their C-banding pattern and nucleolus organizer regions (NORs). In these species, the constitutive heterochromatin occurs in the pericentromeric region of all chromosomes, and additionally in the interstitial regions of the sex chro- mosomes. The use of silver staining to reveal the location of NORs on the chromosomes of these species have shown similar patterns to those produced by C-banding (Virkki, 1983; Virkki and Denton, 1987), which probably does not correspond to the NORs pattern. Consequently, the NORs pattern has not yet been established for this group. Considering that little karyotype information exists in the literature on Oedionychina species (1 Disonychina and 38 Oedionychina) from the Brazilian fauna, the purpose of this work was to characterize for the first time the karyo- type of Paranaita opima in relation to its diploid number, chromosomal morphology, type of sex determination sys- tem, constitutive heterochromatin distribution pattern, and NOR-bearing chromosomes. Material and Methods We analyzed 17 Paranaita opima (Germar, 1824) specimens (Figure 1), of which 5 were male embryos and 12 were adults (9 males and 3 females). The adults were collected at the Experimental Garden of the Instituto de Biociências, Universidade Estadual Paulista (UNESP), Rio Claro (22°24’ S, 47°33’ W), São Paulo, Brazil and the em- bryos were obtained in the laboratory from females natu- rally fertilized in the field and in captivity. The mitotic chromosomes were obtained from the embryos and also from the gonads of adult P. opima. The embryos and adult gonads were removed in physiological saline solution for insects and were processed according to the methodology of Webb et al. (1978). The testes, but not the embryos, were submitted to a hypotonic treatment (tap water) for 3 min and then testes and embryos were fixed in Carnoy I mixture (3:1 methanol: acetic acid) for 30 min and then transferred to a drop of 45% (w/v) aqueous acetic acid on a microscope slide and the material macerated to form a cell suspension, the slides being dried at 35-40 °C on a hot-plate, stained with 3% (w/v) Giemsa in phosphate buffer (pH 6.8) for 12 min, rinsed in distilled water and air-dried. The slides were examined by bright-field optical microscopy using a 100x oil-immersion objective fitted to a Zeiss optical photomicroscope and Kodak Imagelink HQ Microfilm. The C-banding (Sumner, 1972) and silver nitrate nu- cleolar organizer region (Ag-NOR) staining (Howell and Black, 1980) were carried out on the Giemsa-stained chro- mosome preparations described above after removing the immersion oil with Xylene. Triple fluorescent-staining was applied using the GC-specific fluorochrome chromomycin A3 (CMA3) and the AT-specific fluorochrome 4’-6-diami- dino-2-phenylindole (DAPI), both combined with the distamycin A (DA) counterstain using the technique de- scribed by Schweizer (1980). Routine cytological analyses were carried out as de- scribed above and fluorochrome analyses were performed using an Olympus BX50 photomicroscope fitted with fil- ters specific for the DAPI and CMA3 fluorochromes, pho- tomicrographs being made using Kodak T-Max Film. Chromosome morphology was characterized as described by Levan et al. (1964). Results Standard staining with Giemsa The P. opima mitotic metaphases showed a chromo- some complement of 2n = 22 (20+XY) for males and 2n = 22 (20+XX) in females. The karyotype obtained from embryos and gonial metaphases revealed that the diploid complement consists of three submetacentric chromoso- mes (pairs 1, 2 and 5), seven metacentric autosomes (pairs 3, 4 and 6 to 10) and extremely large metacentric X and Y sex chromosomes (Figure 2). 476 Almeida et al. Figure 1 - Specimens of Paranaita opima. A. Female. B. Male. Magnifi- cation = 5.2X. In most of the embryonic, spermatogonial, and oogo- nial mitotic metaphases, the pericentromeric region of all the chromosomes was negative heteropycnotic. Additio- nally, in these mitotic metaphases, a prominent secondary constriction was observed at the interstitial region of the short-arm of pair 6, and the short-arm terminal region of this pair also showed negatively heteropycnotic (Figures 2, 3A, 4A, 6A and 6F). C-banding In mitotic metaphases, all the chromosomes, includ- ing the sex chromosomes, showed the occurrence of strong- ly labeled constitutive heterochromatin in the centromeric region (Figure 3B). In pair 6, the centromeric constitutive heterochromatin extended to the short arm until the second- ary constriction (Figures 3B, 6B and 6G). In some embryonic metaphases, the chromosomes of which were more distended, the Y chromosome showed ad- ditional constitutive heterochromatin on the interstitial re- gion of one of the chromosome arms (Figure 3B). In addition, other differential, but not well-defined, marks were visible on the arms of the sex chromosomes. Ag-NOR staining Some Giemsa-stained gonial metaphase cells submit- ted to Ag-NOR staining showed the NORs at the secondary constriction region of pair 6 (Figures 4B, 6C and 6H). Triple fluorochrome staining Most embryonic mitotic metaphases showed chromo- somes with CMA3-negative centromeric regions and ho- mogeneously stained arms (Figure 5A), the exception being pair 6 which showed a CMA3-positive chromosomal region on the short arm which extended from the proximal to the interstitial region (Figure 5A) and was partially coin- cident with the secondary constriction and the C-band (Fig- ures 6A, 6B, 6D, 6F, 6G and 6I). Analysis with DAPI filter revealed the presence of fluorescent AT bands at the centromeric region of all chromosomes of the complement, including the sex chromosomes (Figure 5B). Pair 6 showed DAPI-positive fluorescence at the terminal region of the short arm (Figures 5B, 6E and 6J) in addition to that on the centromeric region and a DAPI-negative region inserted between the centromeric and telomeric DAPI-positive re- gions, including all positive CMA3 region. Discussion The basic karyotype characteristics of P. opima, such as diploid number, chromosomal morphology, type of sex determination system, and C-banding pattern are similar to those described for other Oedionychina species (Virkki, 1961, 1964, 1970; Smith and Virkki, 1978). The P. opima chromosomal number of 2n = 22 could have been derived from 2n = 24, which has been proposed as ancestral for Chrysomelidae by Virkki (1970) and Smith and Virkki (1978), and may have resulted from fusion Karyotype characterization of Paranaita opima 477 Figure 2 - Giemsa-stained Paranaita opima male embryo with 2n = 22 (20+XY) karyotype. Note the secondary constriction (arrow) and the neg- ative heteropycnosis (arrowhead) on pair 6 and the extremely large sex chromosomes. Bar = 10 �m. Figure 3 - Male Paranaita opima embryo mitotic metaphase with 2n = 22 (20+XY). A. Standard Giemsa staining. B. The same cell as shown in A submitted to Giemsa staining followed by C-banding and showing centro- meric constitutive heterochromatin blocks on all chromosomes. Arrows indicate the centromeric region of some chromosomes. The arrowhead points to one of the interstitial C-bands on the sex chromosome. Bar = 10 �m. events involving only autosomes or autosomes and sex chromosomes. Similar proposals have been made by Virkki (1970) and Smith and Virkki (1978) for the majority of the Oedionychina species that possesses 2n = 22 chromoso- mes. The presence of metacentric chromosomes in P. opima is in agreement with the condition that prevails in the most current Coleoptera species and that is considered by Smith and Virkki (1978) as a basic and ancestral karyotypic characteristic. Our results show that P. opima has similar-sized X and Y sex chromosomes, a characteristic that has also been found in two other Oedionychina species (Alagoasa (under Oedionychus) acutangula and Alagoasa extrema), whereas the X and y chromosomes of the majority of the species of this subtribe, including Paranaita bilimbata, are different in size. In general, Oedionychina X and y chromosomes are asynaptic during meiosis but present regular segregation (Virkki, 1968). 478 Almeida et al. Figure 4 - Adult Paranaita opima oogonial metaphase with 2n = 22 (20+XX). A. Giemsa staining, showing chromosomal elements of pair 6 (arrowhead). B. The same cell as shown in A, with Giemsa and Ag-NOR sequential staining showing NORs on pair 6 (arrowhead). Bar = 10 �m. Figure 5 - Male Paranaita opima embryo mitotic metaphase stained with CMA3, DA and DAPI and showing 2n = 22 (20+XY). A. Chromomycin A3 staining, showing the CMA3-negative centromeric region (arrowhead) of some chromosomes and the positive CMA3 labeling at the interstitial region of the short arm of pair 6 (arrow). B. DAPI staining showing the DAPI-positive centromeric region (arrowhead) of some chromosomes, the negative DAPI interstitial region of the short arm of pair 6 and the DAPI-positive terminal region of the short arm of pair 6 (arrow). Bar = 10 �m. The C-banding pattern of P. opima was coincident with those described by Virkki (1983) for some Oedionychina species (Omophoita annularis, Omphoita personata, Omophoita octoguttata and Alagoasa januaria) and the P. opima sex chromosomes also exhibit additional C-bands on the interstitial region of the chromosome arms, varying in number, position and intensity according to the degree of chromosome distention, these bands being simi- lar to those described in O. annularis, O. personata, O. octoguttata and A. januaria. The presence of interstitial C-bands on the large sex chromosomes of Oedionychina species could be related to the origin of this sex determination system in that these bands could represent heterochromatic material remaining from ancient autosomes incorporated into the original sex determination system of the XY type, or from ancestral y chromosomes belonging to the Xny-type sex determination system. According to Virkki (1970), the large and asynap- tic sex chromosomes in Oedionychina could be derived from translocations of the X and Y chromosomes to a large pair of autosomes, such as those found in Phyllotrupes (Alticinae, Systenini), or from fusions among y chromo- somes belonging to the Xny-type multiple sex determina- tion system found in many Disonychina species, depending on the evolutionary relationship between the subtribes. We found that in P. opima the NORs occur on pair 6, remarkably different from the pattern described for P. bilimbata and other Oedionychina species (O. annularis, O. personata, O. octoguttata, A. januaria, Alagoasa bicolor, Omophoita cyanipennis and Omophoita albicollis), which is characterized by multiple marks on the y chromosome (Virkki, 1983; Virkki and Denton, 1987). According to Virkki (1983), this pattern of multiple marks on the y chromosome could correspond to a peculiar type of chromatin and not to NORs. In other insect groups, the use of silver nitrate impregnation to detect NOR-bearing chro- mosomes has also shown special types of constitutive heterochromatin and other chromosome structures (Rufas et al., 1983; Cella and Ferreira, 1991). In the family Chrysomelidae, NORs have been estab- lished for a few species (Botanochara angulata, Calligrapha polyspila, Chelymorpha variabilis, Chrysolina americana, Chrysolina bankii and Zatrephrina meticulosa) and are located on one autosomal pair, some- times at the secondary constriction and sometimes not. In some of these species the NOR-bearing autosomal pair has been identified as either the largest pair or pair 5 (Posti- glioni et al., 1990, 1991; Postiglioni and Brum-Zorrilla, 1988; Petitpierre, 1996). The distribution of NORs has only been studied in a few Coleoptera species, but in families such as the Curculionidae and Tenebrionidae the NORs oc- cur on autosomes and on sex chromosomes, while in the Cicindelidae and Coccinellidae they occur on autosomes or on sex chromosomes (Drets et al., 1983; Virkki et al., 1991; Galián et al., 1995; Juan et al., 1993; Maffei et al., 2001). Our results obtained with the CMA3 and DAPI fluo- rochromes showed that the centromeric region of all P. opima chromosomes is AT-DNA-rich. The presence of DNA with highly repeated AT base sequences in the cen- tromeric region has also been found in the chromosomes of some Tenebrionidae (Juan et al., 1991; Plohl et al., 1993). In P. opima, the secondary constriction region on pair 6 appeared to include a GC-rich portion in addition to the NOR because one portion of this region was CMA3 positive while the other was silver impregnated and, moreover, all this region was negative for both C-banding and DAPI. On the other hand, the short arm positive DAPI terminal region of pair 6 was CMA3-negative, confirming that this region contains AT base pair sequences. Considering all these re- sults, the NORs on pair 6 seems to be flanked by a special type of heterochromatin, rich in AT base sequences at the terminal side and rich in GC base sequences at the proximal side. These special types of heterochromatin flanking the NORs were not shown by the usual C-banding technique. The presence of constitutive heterochromatin flanking or inserting NORs has been found in the chromosomes of sev- eral different groups of animals (Cabrero et al., 1986; King et al., 1990; Pendás et al., 1993; Silva et al., 2000; Vicari et al., 2003, 2005). The presence of this NOR-associated heterochromatin can restrict genetic recombination in the adjacent region, avoiding the formation of chiasmata or in- ducing their formation in other chromosome regions (John and King, 1982, 1985) and also can alter the genetic expres- sion of the NORs (Arnold and Shaw, 1985) or can represent breakpoints that facilitate the dispersion of the NORs (Mo- reira-Filho et al., 1984; Reed Phillips, 1995). Our results offer important insights into the karyotype characteristics of P. opima, which may be useful in eluci- Karyotype characterization of Paranaita opima 479 Figure 6 - Paranaita opima pair 6 chromosomal elements submitted to different staining techniques (A-E) and their respective schematic repre- sentation (F-J). A and F. Giemsa staining showing secondary constriction (arrow) and negative heteropycnosis (arrowhead) at the short arm terminal region. B and G. C-banding showing a large constitutive heterochromatic block at the pericentromeric region. C and H. Ag-NORs at the secondary constriction. D and I. CMA3 and DA staining showing the GC-rich DNA at the short arm interstitial region. E and J. DA and DAPI staining showing the AT-rich DNA at the centromere and short arm terminal region. dating relationships between species of the subtribe Oedionychina. 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