Meiosis and fluorescent banding in Edessa meditabunda and E. rufomarginata (Heteroptera: Pentatomidae: Edessinae)

Species of Pentatomidae are cytogenetically characterized by the presence of holokinetic chromosomes, a pre-reductional type of meiosis, and a great constancy not only in chromosome number (2n = 14 in 85% of the 250 species analyzed) but also in the sex chromosome determining system (XY/XX). Edessa meditabunda and E. rufomarginata males have 2 n = 1 4 = 1 2 + XY, and both species present small telomeric positively heteropycnotic bands which are DAPI and CMA bright. In E. meditabunda the NOR region is clearly revealed at the telomeric region of the largest autosomal pair by silver staining and CMA banding. Meiotic behaviour of both species follows the general pat­ tern of the order: autosomes divide pre-reductionally, sex chromosomes are achiasmatic and divide postreductionally, and at both metaphase plates the autosomes become arranged in a circle with the sex chromosomes lying at its center. In E. meditabunda, how­ ever, the larger sex chromosome is generally observed at metaphase I forming part of the ring of autosomal bivalents. Bivalents with two chiasmata are frequently observed in E. meditabunda and E. rufomarginata', mean chiasma frequency (6.45 and 6.26, respec­ tively) differ significantly between both species, but differences between populations within each species are not significant. The metaphase plate arrangement of autosomes and sex chromosomes is rather constant in Heteroptera. However, our results in E. meditabunda together with previous reports in other species of the order led us to suggest that the metaphase plate arrangement is more liable to variation at the first meiotic division than at the second one, when it is almost constant. The presence of ring bivalents in both species here analyzed constitutes further evidence against the previous statement of only one chiasma per bivalent in Heteroptera.

One of the most important cytogenetic characteristics of the order Heteroptera is the holokinetic nature of its chro mosomes, in contrast to the monocentric chromosomes (i.e., with a localized centromere) present in most organ isms (Ueshima, 1979).Within the Pentatomidae 250 spe cies belonging to six subfamilies have been cyto genetically analyzed, and a great constancy in chromosome number (2n = 14 in 85% of the species) and the sex chromosome determining system (XY/XX in 99% of the species) has been reported (Dey & Wangdi, 1988;Manna & Deb-Mallick, 1981;Nuamah, 1982;Rebagliati, 2000;Rebagliati et al., 2001;Sathapathy & Patnaik, 1988;Ueshima, 1979).The subfamily Edessinae is one of the less studied, 11 out of the 13 species cytogenetically analyzed belong to the genus Edessa, and all of them have 2n = 14, n=6 + XY (Table 1).
In the present contribution, male karyotype, heterochro matin composition and distribution (DAPI-CMA banding), and meiotic behaviour of E. meditabunda and E. rufomarginata are described and compared; further more, the silver staining technique was applied to E. meditabunda in order to reveal the location of the nucleolus organizing region.Finally, chiasma frequency within and between the species is also analyzed.

MATERIAL AND METHODS
The present study was performed on adult males of E. meditabunda from Macomita (8 males) (Tucumán Province); Gilbert (4 males), Gualeguaychú (3 males), Almada (4 males), and Colón (4 males) (Entre Ríos Province); Hughes (1 male) (Santa Fe Province); and males of E. rufomarginata from Cuchilla de las Aguilas (12 males) (Benito Juárez, Buenos Aires The specimens were fixed in 3:1 absolute ethanol: glacial acetic acid; later, gonads were dissected out and kept in 70% ethanol at 4°C.Slides were made by the squash method in iron propionic haematoxylin or acetic carmin.Fluorescent staining with the GC specific chromomycin A3 (CMA3) and AT spe cific 4'6-diamidino-2-phenylindole (DAPI) was performed on unstained slides.A piece of gonad was squashed in 45% acetic acid, the coverslip was then removed by the dry-ice method, and the slide was air-dried.Afterwards, the slides were incubated in methanol for 2 hs and then air-dried.One drop of a solution of 0.4 pg/ml DAPI in Mac Ilvaine's buffer pH 7 (0.1 M citric acid, 0.2 M Na2HPO4) was placed on each slide, covered with a cov erslip, and incubated for 20 min at room temperature.The slides were then rinsed with distilled water, Mac Ilvaine's buffer and distilled water, and air-dried.Afterwards, one drop of a solution of 0.6 mg/ml CMA3 in Mac Ilvaine's buffer with 10 mM MgCl2.6H2O was placed on each slide, covered with a coverslip, and incubated for 1 hr at room temperature.The slides were then rinsed with distilled water, Mac Ilvaine's buffer and dis tilled water, and air-dried.Slides were mounted in a drop of glycerol-Mac Ilvaine's buffer with 10 mM MgCl2.6H2O, and stored at 37°C for 72 hs before microscopic analysis.The incu bation and air-drying steps were performed in the dark.Fluores cence analysis was performed in a Leica Wild MPS52 fluores cence microscope with Leica appropriate filters.
The silver staining technique was performed on E. meditabunda slides as previously described (Papeschi, 1995).
Chiasma frequency of cells at diakinesis and metaphase I was compared by means of a three-level nested ANOVA (Sokal & Rohlf, 1995).

Meiotic analysis
Spermatogonial cells of E. meditabunda show 2n = 14 = 12 + XY chromosomes without primary constrictions (holokinetic chromosomes) (Fig. 1A).At early meiotic prophase sex univalents X and Y are positively heteropycnotic.At pachytene they are frequently placed close to each other, and the nucleolus can be observed associated x ± SD = 6.24 ± 0.027 to the telomeric region of a large autosomal bivalent.At the diffuse stage, the autosomal bivalents decondense completely while the sex chromosomes continue associ ated and positively heteropycnotic (Fig. IB).At diplotene the autosomes recondense, and their telomeric regions are slightly positively heteropycnotic.After late diplotene (Fig. 1C) and at diakinesis (Fig. ID) the X and Y chro mosomes become isopycnotic.From late diakinesis until anaphase II the larger sex chromosome continues isopyc notic with the autosomal bivalents while the smaller one is negatively heteropycnotic (Fig. 1E-J).At metaphase I the autosomal bivalents arrange in a circle with the sex univalents placed at different positions: both the X and the Y lying at the center of the ring (as is usual in Heteroptera) (28%, from a total of 110 cells) (Fig. 1F); or the smaller sex chromosome at the center of the ring and the larger forming part of it (72%) (Fig. 1G).At anaphase I the autosomal bivalents divide reductionally and the sex univalents, equationally (Fig. 1H).During the second meiotic division, the metaphase II arrangement is always the same: the sex chromosomes associate end-to-end forming a pseudobivalent (touch-and-go pairing), and lie at the center of the ring of autosomes (Fig. 1I).At ana phase II the sex chromosomes X and Y segregate to opposite poles (post-reductional division) (Fig. 1J).No In spermatogonial cells of E. rufomarginata a diploid number of 14 (2n = 12 + XY) is observed, and most chro mosomes present positively heteropycnotic blocks at telomeric position (Fig. 2A-B).At pachytene, sex chromosomes are frequently associated and are posi tively heteropycnotic.During the diffuse stage, autosomal bivalents decondense while the X and Y continue associ ated and positively heteropycnotic (Fig. 2C).At diplotene the nucleolus can be observed associated to the telomeric region of an autosomal bivalent (Fig. 2D).From early prophase until this stage telomeric regions of autosomal bivalents are positively heteropycnotic (Fig. 2C-D).At diakinesis, sex chromosomes become isopycnotic and lie separated (Fig. 2E).At metaphase I autosomal bivalents arrange in a circle with the X and Y univalents at its center (Fig. 2F-G); the first meiotic division is reductional for the autosomes and equational for the sex chromo somes.At metaphase II the XY pseudobivalent lies at the center of the ring of autosomes (Fig. 2H).
In both E. meditabunda and E. rufomarginata, auto somes show a gradation of sizes, and the sex chromo somes are the smallest of the complement (Fig. 3).The X and Y chromosomes are unequal in size, and this differ- ence is more conspicuous in E. meditabunda than in E. rufomarginata.

DAPI and CMA banding
DAPI and CMA banding in E. meditabunda reveals that sex chromosomes are DAPI bright and CMA bright during early prophase until diplotene (Fig. 5A-B).At metaphase I the smaller sex univalent is DAPI dull while the larger is as uniformly fluorescent as the autosomes (Fig. 5C, 5E).This difference continues until metaphase II (Fig. 5F).Autosomal bivalents show small DAPI bright and CMA bright bands at telomeric regions, while a con spicuous CMA bright and DAPI dull band is detected at a pair of telomeric regions of the largest autosomal pair (Fig. 5A, 5B, 5D).Similarly, in E. rufomarginata, sex chromosomes are DAPI bright during early meiotic pro phase and the telomeric regions of autosomal bivalents are slightly DAPI bright and CMA bright.At both mei otic metaphases the larger sex chromosome is as DAPI and CMA bright as the autosomes, while the smaller one is as fluorescent with CMA as the autosomes but slightly less DAPI bright (Fig. 5G-H).

DISCUSSION
Within the subfamily Edessinae thirteen species have been cytogenetically analyzed, of which eleven belonged to the genus Edessa (Table 1).Edessa rufomarginata had been previously studied by Schrader (1941a), while the cytogenetics of E. meditabunda is described in the pre sent paper for the first time.
In the order Heteroptera, chromosome arrangement at both meiotic metaphase plates is very specific and precise; although there are variations among different families, it is rather constant within each one (Ueshima, 1979).The most common pattern is the arrangement of autosomes in a circle with the sex chromosomes (either as univalents at metaphase I or associated in a pseudobiva lent at metaphase II) lying at its center.Edessa rufomarginata follows this chromosome arrangement.On the other hand, some irregularities have been observed in metaphase I plates of E. meditabunda.The typical pattern is observed in some cells, but in most of them (72%) the larger sex univalent forms part of the circle of autosomal bivalents while the smaller one lies at its center.Schrader (1941a) also described in E. irrorata that at metaphase I all chromosomes are arranged in a circle, but in many cells one or two chromosomes (not necessarily the sex univalents) lie at its center.This alteration of the regular pattern in E. meditabunda and E. irrorata, together with previous reports in species of Belostoma (Belostomatidae, Heteroptera) (Papeschi, 1992), give further support to the suggestion that the spatial arrangement of the different chromosome kinds is more liable to variation at meta phase I than at metaphase II, when it is almost invariable.
Bivalents with two chiasmata are frequently observed both in E. meditabunda and E. rufomarginata.The analysis of mean chiasma frequency reveals that in 50% of the cells of E. meditabunda and 25% of E. rufomargi nata a ring bivalent is present.These results constitute additional evidence that support our suggestion that ring bivalents are not so rare in Heteroptera as earlier studies proposed (Rebagliati et al., 2001;Ueshima, 1979).
With reference to the heterochromatin content and dis tribution in Edessa species, Schrader (1941a) reported positively heteropycnotic telomeric regions in the auto somes of E. irrorata, E. costalis, and E. rufomarginata.
In the present study, E. rufomarginata and E. meditabunda showed small telomeric positively hetero pycnotic blocks that were DAPI bright and CMA bright until diakinesis.The telomeric localization of heterochro matin has already been described by C banding in many heteropteran species (Panzera et al., 1992(Panzera et al., , 1995;;Papeschi, 1991); the bright fluorescence of these regions in E. meditabunda and E. rufomarginata both with DAPI (which reveals AT-rich DNA) and CMA (which enhances GC-rich DNA) could be explained if these regions were heterochromatin with interspersed AT and GC repeats.The conspicuous CMA bright band detected at the telo meric region of the largest autosomal bivalent in E. meditabunda together with the fact that the nucleolus, as revealed by silver staining, was associated with the telo meric region of this bivalent, led us to conclude that the NOR region is associated with a GC rich band.This cor respondence between CMA positive banding and NOR regions has also been described in the X chromosome of aphids (Bizzaro et al., 1999;Mandrioli et al., 1999).In the present contribution the DAPI and CMA bright fluo rescence of the sex chromosomes of E. meditabunda and E. rufomarginata until diplotene is consistent with the allocycly of these chromosomes during male meiosis, and it probably reflects different degrees of chromatin con densation rather than differences in base composition.From early prophase until diakinesis the X and Y chro mosomes are both more condensed than the autosomes, while at this meiotic stage the autosomal bivalents and the larger sex chromosome are equally condensed.Con versely, the smaller sex chromosome is negatively hetero pycnotic and less bright with the fluorescent dyes from metaphase I until the end of meiosis.The difference in fluorescence and pycnosis of the smaller sex chromosome could be due to a slight decondensation of this chromo some, or as a consequence of its small size and its strong stretching between the poles by the spindle fibers at the metaphase plates.On the other hand, differences in the pycnotic and fluorescent response of the smaller sex chro mosome between E. meditabunda and E. rufomarginata could be explained by differences in the DNA content of these chromosomes.

Fig. 1 .
Fig.1.Edessa meditabunda (2n = 14,n = 6 + XY).A -spermatogonial prometaphase; B -diffuse stage; C -early diakinesis; D -middle diakinesis; E -late diakinesis; F -meta phase I: both the X and the Y lie at the center of the ring of autosomal bivalents; G -metaphase I: the smaller sex chromosome lies at the center of the ring of the autosomal bivalents, while the larger one forms part of it; H -anaphase I; I -metaphase II: the XY pseudobivalent lies at the center of the ring of autosomes; J -anaphase II.Arrowheads point to ring bivalents, arrows show sex chromosomes.N = nucleolus.Bar =10 pm.

Fig. 4 .
Fig. 4. Ag-staining in Edessa meditabunda.A -Mitotic prometaphase; B-C -diplotene; sex chromo somes are separated in (B) and associated in (C); D -detail of NOR bivalent.Nucleoli are connected to the telomeric regions of the largest auto somal pair.N = nucleolus.Bar =10 pm.

Fig. 5 .
Fig. 5. DAPI (blue) and CMA (green) banding in E. meditabunda (A-F) and DAPI banding in E. rufomarginata (G-H).E. meditabunda: A-B -diplotene; C-E -metaphase I;F-metaphase II.E. rufomarginata: G -metaphase I;H-metaphase II.Arrowheads point to sex chromosomes.Arrows in B-D show CMA bright band at the telomeric region of one large autosomal bivalent.Bar =10 pm.All figures with the same magnification except E (Bar =10 pm).

Table 1 .
Male diploid and haploid chromosome numbers of species ofEdessinae.

Table 2 .
Number of chiasmata (q)and mean chiasma fre quencies at diakinesis-metaphase I in specimens of Edessa meditabunda from Macomita (M) and Gilbert (G) and of E. rufomarginata from two samples from Benito Juárez (B1 and B2).

Table 3 .
Analysis of variance of mean chiasma frequency at diakinesis-metaphase I in specimens of Edessa meditabunda and of E. rufomarginata.