Karyotype Characterization of Planthopper Species Hysteropterum Albaceticum Dlabola, 1983 and Agalmatium Bilobum (Fieber, 1877) (Homoptera: Auchenorrhyncha
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© Comparative Cytogenetics, 2009 . Vol. 3, No. 2, P. 111-123. ISSN 1993-0771 (Print), ISSN 1993-078X (Online) Karyotype characterization of planthopper species Hysteropterum albaceticum Dlabola, 1983 and Agalmatium bilobum (Fieber, 1877) (Homoptera: Auchenorrhyncha: 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 Animals, 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 (Issidae). 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 Insects 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- Comparative Cytogenetics Comp. Cytogenet., 2009 3(2) 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- Comparative Comp. Cytogenet., 2009 3(2) Cytogenetics 114 V.G. Kuznetsova et al.