© Comparative Cytogenetics, 2009 . Vol. 3, No. 2, P. 111-123. ISSN 1993-0771 (Print), ISSN 1993-078X (Online)

Karyotype characterization of species Hysteropterum albaceticum Dlabola, 1983 and Agalmatium bilobum (Fieber, 1877) (Homoptera: :

Issidae) using AgNOR-, C- and DAPI/CMA3 -banding techniques

V.G. Kuznetsova1, A. Maryańska-Nadachowska2, S. Nokkala3

1Zoological Institute, Russian Academy of Sciences, Universitetskaya nab. 1, St. Pe- tersburg 199034, Russia; 2Institute of Systematics and Evolution of , Pol- ish Academy of Sciences, Sławkowska 17, Kraków 31-016, Poland; 3Laboratory of Genetics, Department of Biology, University of Turku, FIN-20014 Turku, Finland. E-mails: [email protected], [email protected], 3seppo.nokkala@utu.fi

Abstract. Males of Hysteropterum albaceticum Dlabola, 1983 and Agalmatium bi- lobum (Fieber, 1877) display a chromosomal complement of 2n = 26 + X, which is a basic one of the tribe Issini (). In the present study, silver staining, C-banding and

a base specifi c CMA3 -and DAPI-banding were used with the aim of identifying pos- sible cytogenetic markers and distinguishing between karyotypes with the same chro- mosome number and no detectable inter-species differences in karyotype structure. We characterized the species studied in terms of the distribution and molecular structure of C-heterochromatin regions and the location of nucleolus organizer regions (NORs). The species are shown to differ considerably in the amount of heterochromatin, its

distribution pattern along the karyotypes and its stain ability with DAPI and CMA3. Key words: Hysteropterum albaceticum, Agalmatium bilobum, Homoptera, Auche-

norrhyncha, Issidae, NORs, C-heterochromatin, DAPI/CMA3 -staining, chiasmata, polymorphism.

INTRODUCTION exceptions are the investigation of Tibicen The chromosomes of Auchenorrhyncha, bihamatus (Motschulsky, 1861) and Platy- as well as of all other Homoptera groups, pleura kuroiwai Matsumura, 1917 (the family are holokinetic (Halkka, 1959). These chro- Cicadidae) involving C-banding (Perepelov mosomes are characterized by the lack of a et al., 2002), and that of Philaenus spumar- defi ned centromeric locus (localized centro- ius (Linnaeus, 1758) and Ph. arslani Abdul- mere), which makes the identifi cation of in- Nour & Lahoud, 1996 (Aphrophoridae) per- dividual chromosomal pairs and detection of formed by means of a number of differential chromosomal rearrangements in karyotypes staining techniques (Kuznetsova et al., 2003; very diffi cult. Most cytogenetic studies on the Maryańska-Nadachowska et al., 2008). The Auchenorrhyncha were performed using stan- techniques applied characteristically high- dard chromosome staining techniques. The light specifi c chromosomal regions, such as

http://pensoftonline.net/compcytogen Comparative Cytogenetics doi: 10.3897/compcytogen.v3i2.18 112 V.G. Kuznetsova et al. the nucleolus organizer regions (NORs) and advanced techniques are necessary for an ad- C- heterochromatin blocks containing late- equate cytogenetic characterization of this replicating repetitive DNA. Moreover, in both group. studies on the Philaenus Stål, 1864, the DNA In the present study, we have characterized binding fl uorochromes CMA3 and DAPI were the karyotype of two Issini species, Hysterop- used to reveal whether the heterochromatin is terum albaceticum Dlabola, 1983 and Agal- enriched in GC or AT bases. The above meth- matium bilobum (Fieber, 1877), using silver odologies have provided cytogenetic markers staining, C-banding and base specifi c CMA3- for understanding chromosome diversity and and DAPI-banding, with the aim of identify- suggesting mechanisms of chromosome rear- ing possible cytogenetic markers and distin- rangements in the genus Philaenus (Kuznetso- guishing between karyotypes with the same va et al., 2003; Maryańska-Nadachowska et chromosome number 2n = 26 + X. As a result al., 2008). we report here for the fi rst time the distribu- The currently available cytogenetic evi- tion and molecular structure of C-heterochro- dence on the world-wide planthopper family matin regions and the location of nucleolus or- Issidae Spinola, 1839 (Fulgoroidea) is restrict- ganizer sites (NORs) in the karyotypes of the ed to 22 species and 15 genera, all but one spe- family Issidae. cies belonging to the tribe Issini Spinola, 1839 (see Maryańska-Nadachowska et al., 2006 and MATERIAL AND METHODS references therein). It is generally assumed that holokinetic chromosomes facilitate karyotype We used adult males of Hysteropterum evolution through fi ssion and fusion of chro- albaceticum collected in Spain and those of mosomes (White, 1973), however the tribe Agalmatium bilobum collected in Greece and Issini seems to demonstrate the considerable Italy in 2005 (Maryańska-Nadachowska et al., karyotypic uniformity, with 2n = 26 + X en- 2006). Freshly caught specimens were fi xed in countered in all but two of the species studied. 3:1 ethanol/acetic acid. This suggests an old and single origin for this karyotype pattern in an ancestor of this tribe. It Chromosome preparations is worth noting that this chromosome comple- All chromosome preparations were from ment matches also a putative ancestral state in testes. Each testis consisted of several long the Fulgoroidea as a whole (Kuznetsova et al., fusiform follicles, 10 in H. albaceticum while 1998; Maryańska-Nadachowska et al., 2006). 11 in A. bilobum. Follicles were dissected out The basic chromosome complement of the in a drop of 45% acetic acid and squashed. Issini looks uniform in rough morphology in Then the preparations were frozen on dry ice different species, with similar size differences and, after removal of coverslips, dehydrated between chromosomes and gaps, probably the in freshly prepared 3:1 ethanol/acetic acid for sites of NOR, visible in some cases in the larg- 20 min and air-dried. The preparations were est pair of autosomes. fi rst analyzed with phase contrast microscope Thus the evidence obtained so far by a rou- at 400 x. The best chromosome spreads were tine cytogenetic technique allows the sugges- used for different kinds of staining. tion that chromosome evolution did not play Standard staining an important role in species diversifi cation of For standard staining the method of Groze- the tribe Issini. It is clear however that more va and Nokkala (1996) was used. The prepara-

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Karyotype characterization of Issidae species 113 tions were fi rst subjected to hydrolysis in 1 N All the preparations were analyzed with HCl at 60° C for 7 min and stained in Schiff’s the aid of a microscope Leica MM 4000 at reagent for 20 min. After rinsing thoroughly 1000 x or (the fl uorochrome-labeled prepara- in distilled water, the preparations were addi- tions) a fl uorescence microscope Dialux 22 at tionally stained in 4% Giemsa in Sorensen’s 1000 x, and the photomicrographs taken using buffer pH 6.8 for 20 min, rinsed with distilled a Camera Nikon DS-U1. water, air-dried and mounted in Entellan. C-banding RESULTS For C-banding the method of Sumner Hysteropterum albaceticum Dlabola, 1983; (1972) was used. The treatment was carried 2n ♂ = 26 + X out using 0.2 N HCl at room temperature In the present study, the male chromosomal for 30 min, a 7-8 min treatment in saturated formula of this species, n = 13 + X (sex determi- nation is X0), reported earlier by Maryańska- Ba(OH)2 at room temperature and then a in- cubation in 2xSSC at 60°C for 1 h. After this, Nadachowska with co-authors (2006), was the preparations, fi nally stained in 4% Giemsa confi rmed. Unlike the mentioned publication, diluted in Sorensen buffer for 10-15 min with in which the karyotype was deduced solely excessive staining briefl y rinsed in tap water, from one stage, the spermatocyte metaphase were mounted in Entellan. I, we followed a number of consecutive stages of male meiosis and applied the above-listed AgNOR-staining cytogenetic techniques all allowing an ex- For silver impregnation the method of tended characterization of the chromosome Howell and Black (1980) was used. Slides complement of this species (Figs 1-14). None were incubated for 6-9 min at 60°C with 60 μl of the specimens analyzed showed spermato- of AgNO 0.5 g/ml and 30 μl of a 2% gelatin 3 gonial mitoses. During meiotic stages, 13 bi- and 1% formic acid solution. The preparations valents and a univalent X chromosome were were rinsed with abundant distilled water and observed; one of the bivalents was noticeably air-dried. larger than the others; the remaining bivalents

CMA3- and DAPI-banding were gradually decreasing in size; and a rather For fl uorochrome application the methods large X was close in size to the large-sized of Schweizer (1976) and Danlon and Magenis half-bivalents. Only in a few preparations, C- (1983) were used with minor modifi cations. banding has induced C-blocks, which were C-banded preparations (without Giemsa) found to be present in the majority of bivalents were stained fi rst with chromomycin antibi- and were of different size and location, both otic CMA3 (5 μg/ml) for 25 min and then with telomeric and interstitial. The largest bivalent DAPI (0.4 μg/ml) for 5 min. To improve the exhibited the most conspicuous blocks, which fl uorochrome staining, 0.5% methanol was in- were always located at only one end of every cluded in the staining solutions (Kuznetsova homologue. Furthermore, this bivalent showed et al., 2001). After staining, the preparations a heteromorphy for the size of C-blocks, i.e., were rinsed in the McIlvaine buffer, pH 7 and they were different in size in the homologues mounted in an anti-fade medium (700 μl of of this pair (Figs 1, 2). This pattern was also glycerol, 300 μl of 10 mM McIlvaine buffer, confi rmed by DAPI staining, which has been pH 7, and 10 mg of N-propyl gallate). operating successfully in this species (Figs 3-

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114 V.G. Kuznetsova et al.

Figs 1-14. Male meiotic chromosomes of Hysteropterum albaceticum. 1, 2 - late diakinesis, C-banded; 3 - pre- meiotic interphase, DAPI-staining showing 7 bright and 2 low-power signals; 4-10 - dilpotene (4, 5) and diakinesis (6-10), DAPI-stained. The largest bivalent shows one (4, 6, 7, 9, 10), two (8) and three (5) chiasmata. Arrows point to interstitial signals in the same middle-sized bivalent (4-7) and to two interstitial signals in one homologue of the largest bivalent (9); 11-12 – diplotene/diakinesis, CMA3-stained. Arrow point to signals in several bivalents (11) and to NOR with a nucleolus in one homologue of the largest bivalent (12); 13, 14 - diplotene, silver impregnation showing nucleolar remnants in one (13) and both (14) homologues of the largest bivalent. Bar = 10 μm.

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Karyotype characterization of Issidae species 115

10). Heterochromatin particles were scattered homologues. This is probably related to the in premeiotic interphase cells and DAPI stain- differential activity of the NORs in different ing has produced approximately 10-12 fl uores- homologues. Of interest is however the loca- cent signals of various sizes and brightness in tion of the silver stained nucleolar material these cells (7 bright and 2 low-power signals clearly at the submedial position rather than are seen in Fig. 3). This staining also revealed at the ends of the chromosomes. The origin of bright signals in the majority of bivalents and these different patterns, if this is due to an in- in the X chromosome in prophase cells; two version fl anking the NOR region or to some or three bivalents only were nearly devoid of other reasons, remained unknown. heterochromatin (Figs 4-10). During diplotene In all the bivalents other than the largest stage, certain of the bivalents displayed DAPI- one, there occurred invariably only one cross- positive signals near the end of chromosomes, over point. In diplotene cells, different lo- more often at only one end of a homologue. calization of chiasmata could be seen in the However in some bivalents, the signals were bivalents: terminal or subterminal in some bi- clearly located interstitially (arrowed in Figs valents while clearly interstitial in 3-4 larger 4, 5, 9). One of these bivalents could be easily bivalents (Figs 4-5). In the largest bivalent, recognized in every diplotene and almost every a chiasma was present at different positions, diakinetic (arrowed in Figs 4-7) nucleus due to medial, subterminal or terminal, in diplotene/ the presence of a bright interstitial signal in its diakinesis cells. In some cells, this bivalent homologues. The largest bivalent was charac- demonstrated the ring shaped formation that teristically marked by bright telomeric signals is indicative of the occurrence of two chias- in both homologues (always present at one end mata (Fig. 8), and in one cell three chiasmata of the chromosome) but in one deplotene/dia- were seen in this bivalent (Fig. 5). The loca- kinesis cell, one of the homologues exhibited tion of C-and DAPI-positive signals served to two signals of different size and brightness, the identifi cation of the chromosome ends, in both signals having been located interstitially which a chiasma formed in the largest bivalent (Fig. 9). The double-stranded X chromosome and in some other large bivalents. As shown displayed an interstitial signal in all the nuclei in the Figures presented, in these bivalents the studied. Although CMA3 -staining was not as signal could be positioned either at the chro- successful as DAPI-staining, it has also pro- mosomal end far from the chiasma or at the duced clear signals in the majority of biva- chromosomal end near the chiasma. It seems lents, including the largest bivalent (Figs 11, to point to a random chiasma formation in the 12). Thus, the heterochromatin of this species bivalents. However the analysis of 39 larger demonstrated a great affi nity to both fl uoro- bivalents at diplotene/diakinesis stages sug- chromes suggesting that it is equally enriched gested a kind of preferential association be- by AT and GC base pairs. Figure 12 highlights tween these patterns: the chiasma was formed the CMA3 -treated largest bivalent carrying a at a distance of a heterochromatin block in 28 NOR with a nucleolus attached at the end of of these bivalents. one homologue. Silver staining also showed the presence of silver-impregnated fragment- Agalmatium bilobum (Fieber, 1877): 2n♂ ed bodies (nucleolar material) at contact with = 26 + X the largest bivalent, more often with only one Here, the male chromosomal formula of (Fig. 13) while sometimes with both (Fig. 14) this species, n = 13 + X (sex determination

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116 V.G. Kuznetsova et al.

Figs 15-25. Male meiotic chromosomes of Agalmatium bilobum. 15 - Spermatogonial prometaphase, C-band- ed; 16, 17. Diakinesis, C-banded. 18 - premeiotic interphases, DAPI-staining showing 4 bright and 3 low-power signals (top nucleus). 19-22 – diplotene/diakinesis, DAPI-stained. Arrows point to signals in middle-sized biva- lents. 23-25 - Diplotene, silver impregnation showing nucleolar remnants in one (23) and both (24, 25) homo- logues of the largest bivalent. Bar = 10 μm.

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Karyotype characterization of Issidae species 117 is X0), reported earlier by Maryańska-Nada- meiotic stages subjected to DAPI-treatment chowska with co-authors (2006), was con- (Figs 19-22). In diplotene/diakinesis nuclei, a fi rmed. Whereas in the mentioned publication, number of bivalents showed low-power and, this karyotype pattern was deduced from MI then, not necessarily well visible signals, and only, we have studied the chromosome com- only three bivalents, the largest one inclusive, plement of A. bilobum in greater detail with invariably (and sometimes one more; see Fig. the use of mitotic cells as well as meiotic stag- 22) showed bright DAPI-positive signals. One es other than MI, and cytogenetic techniques of these bivalents was the largest one, which such as C-banding (Figs 15-17), DAPI staining exhibited AT-rich regions at one telomere of (Figs 18-22) and silver impregnation (Figs 23- one or more often both homologues. As with 25). One male exhibited spermatogonial mi- H. albaceticum, the longest bivalent appeared toses allowing more precise analysis of chro- as NOR-bearing with numerous nucleolar mosome sizes, compared to meiotic pictures, remnants arranged in this bivalent at a dis- throughout the karyotype. As shown in Fig. tance from the midpoint of one (Fig. 23) or 15, there are 27 chromosomes in a spermato- both (Figs 24, 25) homologues. Each of the gonial prometaphase. All the chromosomes other marker bivalents displayed a brilliant exhibit a well-defi ned holokinetic structure. DAPI-positive signal disposed interstitially By size, we could recognize 5 longer chromo- in one homologue only. All the other biva- somes, approximately 16 medium-sized chro- lents showed either weak or no signals. The X mosomes, and 6 smaller chromosomes. Chro- chromosome was DAPI-negative, suggesting mosomes are undistinguishable within the size that it is defi cient of heterochromatin. Our at- groups. The fi rst group probably includes an tempts to get CMA3-banding have failed in A. X chromosome; however it could not be dis- bilobum. The chromosomes of this species are, tinguished among the chromosomes. therefore, suggested to possess mainly AT-rich Spermatocyte diplotene/diakinesis cells heterochromatin. were made up of 13 bivalents plus the univa- In all diplotene/diakinesis cells investi- lent X chromosome (Figs 16, 17, 19-24). Dur- gated in our material, the bivalents, including ing meiotic stages, one bivalent always stands the largest bivalent, formed each only one chi- out because of its large size; the remaining asma. With the use of DAPI-positive signals bivalents appeared as gradually decreasing in as markers of the chromosomal ends we have size; and the X chromosome was rather large analysed the location of chiasma in the largest and close in size to the large-size half-biva- bivalent in different cells. We have found that lents. C-banding provided conspicuous bands a single chiasma could be formed in any part neither in mitotic nor in meiotic chromosomes of the chromosomes regardless at fi rst glance of this species (Figs 15-17). This result proba- of the heterochromatin location in this biva- bly suggests not only a methodical failure, but lent. However the prevailing pattern found in also a small amount of C-heterochromatin in 10 of 15 diplotene/diakinesis cells investigat- its chromosomes compared to those of H. al- ed was the location of chiasma far from the baceticum. Some of the confi rmatory evidence heterochromatin blocks. comes from A. bilobum premeiotic interphase nuclei, in which usually at most 3-4 bright (plus DISCUSSION 3-4 low-power) DAPI-positive signals were Here we have investigated the role of visible (Fig. 18), as well as from the following chromosomal bandings in distinguishing be-

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118 V.G. Kuznetsova et al. tween karyotypes of two planthopper species, ings have so far been published for the Auche- Hysteropterum albaceticum and Agalmatium norrhyncha. Perepelov with co-authors (2002) bilobum (Auchenorrhyncha, Issidae, Issini), used C-banding to study the karyotypes of exhibiting 2n = 26 +X and a similar general Tibicen bihamatus and Platypleura kuroiwae plan of karyotype structure. Previous cytoge- (the family Cicadidae). The authors found that netic studies of several dozens of species and these phylogenetically remote species, shar- genera of the tribe Issini have shown a strik- ing 2n = 18 + X in males, displayed differ- ing karyotypic uniformity within this group, ences in C-heterochromatin amount and dis- with 2n = 26 +X found in almost all species tribution: in one species the bands were more investigated (Maryańska-Nadachowska et al., abundant and appeared preferentially in the 2006 and references therein; our unpublished terminal chromosome positions, whereas in data). This karyotype has therefore been pro- the other species they exhibited subterminal posed as the basic chromosomal complement and interstitial locations in the chromosomes. for the tribe Issini (Maryańska-Nadachowska Two other publications dealt with the spittle- et al., 2006). It has been generally assumed bug species Phylaenus spumarius and Ph. ar- that holokinetic, or holocentric chromosomes slani (the family Aphrophoridae). These phy- facilitate karyotype evolution by means of dis- logenetically related species were shown to be sociations and fusion of chromosomes (White, similar in having two pairs of NOR-carrying 1973). Although the data on the tribe Issini are chromosomes, GC-rich NORs and dot-like not yet suffi cient to allow defi nite conclusions, C-bands in autosomes while to be different in it is believed that these rearrangements did not having distinct karyotypes, 2n = 22 + X and play an important role in species diversifi ca- 2n = 18 + neo-XY respectively, and dissimilar tion of this group. sex chromosome C- and DAPI staining pat- Outside the same chromosome number, terns (Kuznetsova et al., 2003; Maryańska- the chromosomal complement of the tribe Is- Nadachowska et al., 2008). sini, when studied by conventional cytogenet- A male chromosomal complement, 2n = 26 ic techniques, was shown to display no detect- + X, of H. albaceticum and A. bilobum studied able inter-species differences in chromosome herein agreed with that previously reported size and structure. Chromosome banding tech- for these species by Maryańska-Nadachowska niques are known to produce some important with co-authors (2006). Our karyotype analy- landmarks along the chromosomes that allow sis performed with different meiotic stages (as for the recognition of individual chromosomes well as spermatogonial mitoses of A. bilobum) within a karyotype and identifi cation of spe- revealed also that these species are similar in cifi c regions within individual chromosomes. having one pair of large autosome, 12 pairs of The identifi cation of chromosomal markers in medium-sized autosomes gradually decreas- groups with holokinetic chromosomes is of ing in size, and an X chromosome as one of fundamental importance because of the lack the larger chromosomes in the set. of primary constrictions (the centromeres) in the chromosomes of this type. Consequently NORs rearrangements can not be detected in holoki- The largest pair of autosomes in H. al- netic chromosomes unless different banding baceticum and A. bilobum appeared to be techniques are used. Despite this, as few as NOR-bearing as evidenced by Ag-NOR (in three studies involving chromosomal band- both species) and CMA3 (in H. albaceticum)

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Karyotype characterization of Issidae species 119 bandings. NORs are housed interstitially in matin, which was however clearly more abun- these chromosomes and this location matches dant in H. albaceticum chromosomes. In this the gaps described previously for A. bilobum species, the majority of autosomes and the X- (Maryańska-Nadachowska et al., 2006). It is chromosome are marked by conspicuous C- common knowledge that the nucleolus usually bands of different size and location, terminal, disaggregates during late prophase and reor- subterminal and quite often interstitial. Only ganizes at early telophase. During the diplo- in two or three bivalents, no bands have been tene/diakinesis stages of both species studied detected suggesting that these bivalents are ei- here, nucleolar material (the remnants of ar- ther avoid of heterochromatin or, most likely, gentophilic proteins) was presented as numer- contain little heterochromatin. Several biva- ous silver-impregnated bodies adjacent to the lents, including the largest one, could be easily NOR regions of the chromosomes, the phe- identifi ed in meiotic cells as having very char- nomenon repeatedly described in the subor- acteristic banding patterns. In contrast to what der Heteroptera and known as “the nucleolar is found in H. albaceticum, only the largest and fragmentation phenomenon” (Bardella et al., some three other middle-sized bivalents of A. 2008). In H. albaceticum, the NOR-related bilobum exhibited prominent DAPI-positive heterochromatin appeared to be essentially C-blocks and demonstrated well distinguish-

GC-rich as became evident after CMA3-stain- able banding patterns, and no C-bands have ing. Actually this is true for many other been detected in the X chromosome. groups (Brito et al., 2003; Nechaeva et al., Currently some evidence has been ad- 2004; Severi-Aguiar, 2006; Kuznetsova et al., vanced which characterizes C-heterochroma- 2001, 2007; Diego et al., 2008; Criniti et al., tin of holokinetic chromosomes with respect 2009) and also for two other Auchenorrhyncha to its highly repeated DNA sequences (Biz- species, Ph. spumarius and Ph. arslani, stud- zaro et al., 2000; Kuznetsova et al., 2003; ied in this respect (Kuznetsova et al., 2003; Grozeva et al., 2006; Lanzone, Souza, 2006; Maryańska-Nadachowska et al., 2008). It is Maryańska-Nadachowska et al., 2008). The worthy of note that in these species, as well as results obtained are widely diversifi ed: C-het- in species studied here, the nucleolus organiz- erochromatin composition may at times dem- er regions are placed in autosomes, but both onstrate various patterns of heterogeneity and Ph. spumarius and Ph. arslani were shown to at other times a kind of homogeneity. In H. display two NORs-bearing autosomal pairs. albaceticum, the heterochromatin has shown

C-heterochromatin amount, distribution a great affi nity to DAPI and CMA3-staining and molecular composition suggesting thus that it is enriched by AT and Numerous discussions of C-banding have GC base pairs, i.e. GC-rich clusters are prob- emphasized the small amount of constitutive ably dispersed within AT-rich repeats in its heterochromatin and predominantly terminal heterochromatin. The same is also true for or subterminal location of C-blocks in holoki- NOR-related heterochromatin, which seems netic chromosomes (Collet et al., 1984; Cama- to consist of AT/GC base pair-rich repetitive cho et al., 1985; Blackman, 1987). However DNA sequences in this species. our observations on H. albaceticum and A. The literature on insect constitutive hetero- bilobum are clearly contradictory to this state- chromatin shows that the knowledge of this ment. Both H. albaceticum and A. bilobum type of DNA in insects with holokinetic chro- have shown a great amount of C-heterochro- mosomes is still fragmented and insuffi cient.

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120 V.G. Kuznetsova et al.

In recent years however an increasing quan- tric chromosomes (Schweizer, Loidl, 1987), tity of such data have appeared and shown that is not thus valid for holokinetic chromosomes holokinetic chromosomes can display a great (Mandrioli et al., 2003). deal of C-heterochromatin (Blackman, 1976, 1990; Kuznetsova et al. 1997; Maryańska-Na- Chiasmata dachowska, 1999; Vanzela et al., 2000; Groze- H. albaceticum and A. bilobum showed va, Nokkala, 2001; Golub et al., 2004; Angus chromosomal behavior during spermatocyte et al., 2004; Pérez et al., 2005; Franco et al., meiosis similar to that already reported in oth- 2006; Bressa et al., 2008; Maryańska-Nada- er auchenorrhynchan species (Halkka, 1964; chowska et al., 2008). The species can diverge Kuznetsova et al., 1998, 2009). The bivalents considerably in this pattern: some display a lot most often had a single chiasma but the larger of C-heterochromatin; some show prominent bivalents could sometimes display two chias- C-bands of different size in separate chromo- mata, and the largest bivalent has shown three somes; and in other species C-heterochroma- chiasmata in one cell. The low number of chi- tin appeared to avoid detection due to its pres- asmata (estimated to be 1-2 from cytological ence in small or minute amounts. Actually, analyses) seems to be a standard pattern in ho- this happens also with monocentric groups, lokinetic bivalents (Halkka, 1964). Although in which some species are known to have a the bivalents with multiple chiasmata (up to large amount of C-heterochromatin but some four, inclusive) have been observed in holoki- species not (King, John, 1980; Hoshiba, Imai, netic groups, including the Auchenorrhyncha 1993; Graphodatsky, Fokin, 1993; Swarça et (Kuznetsova et al., 2003, 2009; Maryańska- al., 2003; Rozek et al., 2004). This allows sug- Nadachowska et al., 2008; present paper), gestion that holokinetic chromosomes in fact these observations never advanced beyond differ slightly or not at all in constitutive het- the metaphase I of spermatogenesis and the erochromatin amount from monocentric chro- further fate of cells with multichiasmatic bi- mosomes. The heterochromatin in both holoki- valents remained therefore unknown. Nokkala netic and monocentric chromosomes is known with co-authors (2004) have provided a de- to share a similar molecular composition being tailed analysis of chiasma formation and dis- principally composed of the satellite DNAs tribution in holokinetic bivalents of the psyllid (Mandrioli et al., 2003). These chromosomes species Beopelma foersteri (Flor, 1861) (Ho- are however different in C-heterochromatin moptera, Sternorrhyncha, Psylloidea). These distribution patterns. Monocentric chromo- authors showed that more than two chiasmata somes are known to have pericentromeric and in a holokinetic bivalent will obstruct the reg- telomeric C-bands while very rarely intersti- ular course of meiosis and result in the elimi- tial bands. Holokinetic chromosomes display nation of cells of this type, providing a strong no centromeres and thus lack pericentromeric selection against formation more than two chi- heterochromatin, but telomeric and interstitial asmata in holokinetic bivalents. C-blocks are clearly characteristic of these Heterochromatin is known to suppress re- chromosomes. Thus, a hypothesis of “equilo- combination of chromosomal segments in its cal distribution of heterochromatin”, i.e. the vicinity; this being so, crossing-over rarely oc- tendency of heterochromatin to be located at curs in heterochromatic regions (John, King, similar positions along nonhomologous chro- 1985; Sumner, 1990). We have showed that mosomes (Heitz, 1933) typical for monocen- when only one chiasma was formed in the larg-

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Karyotype characterization of Issidae species 121 er bivalents of H. albaceticum and A. bilobum, following. So far it has been assumed that the chiasma could have medial, subterminal or the auchenorrhynchan tribe Issini is charac- terminal positions in these bivalents in neigh- terized by highly uniform karyotypes giving bouring cells. Using C/DAPI-positive signals no way of distinguishing between the species as markers of the chromosome ends, we have and inferring the chromosome evolution in the analysed the chiasma position variation in the whole group. larger bivalents from both species according Our results showed that even if H. albace- to the heterochromatin distribution per biva- ticum and A. bilobum have karyotypes with lent. We have found that a chiasma could be the same chromosome number and a similar positioned either at the chromosomal end far general plan of structure, they differ consid- from C-heterochromatin block or at the chro- erably in banding patterns; the differences mosomal end adjacent to C-block. It seemed being such as to indicate that many intra- to point to a random chiasma formation in chromosomal rearrangements (although oth- the bivalents. However the aggregate (in both er rearrangements have also been involved) species) analysis of 54 bivalents in diplotene/ are responsible. Moreover, the chromosome diakinesis cells revealed a kind of preferential banding techniques applied have revealed the association between these patterns: in 72% of discriminatory landmarks in the chromosomes the bivalents analysed a chiasma was formed and provided evidence for the existence of at a distance of C-heterochromatin blocks. marked and variable chromosomal polymor- Quite recently, Bressa and co-authors (2008) phism within the studied populations of these have published similar and even more statisti- species. cally plausible results for a bug species Holhy- menia rubiginosa Breddin, 1904 (Heteroptera: ACKNOWLEDGEMENTS Coreidae). In this species, the chiasmata were This work was supported fi nancially by found to occur at any position with reference the Russian Foundation for Basic Research to C-heterochromatin blocks; however in 87% (grants 08-04-00787 and 08-04-10073) and of 203 bivalents investigated a chiasma was by programs of the Presidium of the Russian housed at a point remote from C- blocks. Academy of Sciences “Gene Pools and Ge- Heteromorphism netic Diversity” and “Origin and Evolution of One further observation which clearly the Biosphere” (for VK); and Russian, Finnish needs to be discussed here concerns the fre- and Polish Academies of Sciences. quency of heteromorphisms found in H. al- baceticum and A. bilobum meiosis. These are REFERENCES heteromorhism for NORs of an absence/pres- Angus R.B., Kemeny C.K., Wood E.L. 2004. The C- ence type and that for the presence/absence, banded karyotypes of the four British species of No- size and number of C-heterochromatin blocks tonecta L. (Heteroptera: Notonectidae) // Hereditas. (see Results). However speculations on possi- 140: 134-138. ble explanations would call for more detailed Bardella V.B., Azeredo-Oliveira M.T.V., Tartarotti E. 2008. Cytogenetic analysis in the spermatogen- analysis of these patterns that was beyond the esis of Triatoma melanosoma (Reduviidae; Heter- scope of our present work. optera) // Genet. Mol. Res. 7(2): 326-335. Bizzaro D., Mandrioli M., Zanotti M., Manicardi Conclusions G.C. 2000. Chromosome analysis and molecular The main implication of this study is the characterization of highly repeated DNAs in the

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