Heredity 65 (1990) 157—162 The Genetical Society of Great Britain Received 2 January 1990

Improving beetle karyotype analysis: restriction endonuclease banding of Tenebrio molitor chromosomes

C. Juan*t t Unidadde Genética. Departamento de BiologIa. J. Gosálvezt C-XV, Facuitad de Ciencias, Universidad Autónoma de Madrid, Spain. E. Petitpierre Laboratori de Genètica. Departament de Biologia i C.S., Facultat de Ciències, Universitat de les Tiles Balears, 07071 Palma de Mallorca, Spain.

Fixed mitotic chromosomes of Tenebrio molitor have been analysed by means of C-banding and by digestion with EcoRI and Alu! restriction endonucleases. The chromosomal morphology, ambiguous after conventional staining or C-banding, is much improved by digestion with restriction endonucleases. AluI gives a banding pattern similar to that obtained by C-banding, while EcoRI induces a reverse banding pattern with respect to the previous ones. The EcoRI digestion pattern of chromosomes is probably produced by extraction of a pericentromeric and equilocated highly repeated DNA. In this species, it seems that neither the compaction of the heterochromatin nor the size of the restriction target (six base pairs (bp) for EcoRI) have any effect on the amount of removable DNA. Also, a chromosomal heteromorphism for pair number 9 due to a pericentric inversion has been found in one individual by means of EcoRI treatment, that was cryptic by the other banding techniques.

INTRODUCTION In coleopterans there are relatively few reports of chromosome banding (Ennis, 1974; Angus, Inthe last 20 years banding techniques have pro- 1983; Virkki and Denton, 1987; Juan and Petit- vided an important tool for either the study of pierre, 1989), probably because of the small size chromosome structure and/or identification of of their chromosomes and the poor resolution of individual chromosomes. Recently, restriction longitudinal differentiation obtained with conven- endonucleases (RE) have been used to induce tional techniques. In most cases, the individual chromosome banding by in situ digestion of fixed identification of all chromosomes is difficult due chromatin in a large variety of organisms (Bianchi to the similarities of size and shape. The chromo- and Bianchi, 1987; Lopez-Fernandez et al., 1989), somes of the mealworm beetle Tenebrio molitor are and these simple techniques are very useful for no exception. This species has very similar chromo- detection of specific sequences of DNA, such as somes except for the minute Y chromosome, which for example satellite DNAs (Mezzanotte et al., show very large heterochromatic blocks after C- 1986) or regions which remains cryptic with other banding (Weith, 1985; Juan and Petitpierre, 1989). banding techniques (Gosálvez et a!., 1987; 1989). The large amount of heterochromatin present in Miller et a!. (1983) suggested that the bands pro- each centromere region has been used for two duced by restriction enzymes could also be very different purposes: (i) comparison under electron useful to disentangle the karyotype of organisms microscopy of the euchromatin and constitutive that lack easily banded chromosomes, such as heterochromatin structure (Weith, 1983, 1985) and amphibians, fishes or some plants. In all these (ii) characterization by agarose gel electrophoresis cases a useful level of resolution has been obtained of EcoRI restriction digests and sequencing the after RE-banding (Schmid and Almeida, 1988; satellite DNA thought to be localized in these Lloyd and Thorgaard, 1988; Frediani et a!., 1987), regions (Petitpierre et a!., 1988; Davis and Wyatt, respectively. 1989). The aim of this study is to investigate the action *Presentaddress: School of Biological Sciences, University of of different REs on the chromosomes of this organ- East Anglia, Norwich NR47TJ, U.K. ism, in an attempt to improve the resolution of 158 C. JUAN. J. GOSALVEZ AND E. PETITPIERRE I)'II ii 'ii 4%! •r.3 4 1 2 3 1 a

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Figure1 (a) T molitormalekaryotype with Giemsa staining. (b) C-banded karyotype. Banding patterns produced after digestion with AluI (c) and with EcoRI (d) restriction endonucleases. Bar, 5 m. ENDONUCLEASE BANDING IN TENEBRIO MOLITOR 159 bands produced with other methods. This some and sex chromosome. The distal parts of approach can help obtain a better knowledge of most chromosomal arms appear faintly stained Tenebrio molitor karyotype for analysing poly- (fig. 1(c)). The reverse is seen when treated with morphisms and polytypisms of evolutionary inter- EcoRI, they exhibited median and distal regions est and also be useful in analysing the structural that stain slightly with Giemsa (fig. 1(d)). These characteristics of the huge amount of hetero- regions correspond well with those positive to C- chromatin present in this organism. bands/Alul bands. In fact, the banding pattern obtained with EcoRI is like a reverse C-banding. The digestion of fixed chromosomes with MATERIALAND METHODS EcoRI provides a very good resolution on mitotic metaphsase plates, which is better than that Chromosome preparations obtained with other banding techniques, including Animalswere obtained from a commercial dealer different combinations of specific fluorescent and from a laboratory strain (C.S.I.C, Barcelona). ligands such as chromomycin A3 or diamidino- Male larvae from the third instar were killed and phenylindol (DAPI) (not shown, Juan et aL, in their gonads dissected out. The gonadal tissue was preparation). The use of EcoRI uncovered a immersed in a 005 per cent coichicine solution polymorphism for chromosome 9 which is cryptic for 1 h, hypotonically treated in 0075 M KC1 for with other banding techniques. Indeed, with AluI 5 mm and fixed in fresh ethanol-acetic solution or C-banding the faint staining of the distal (3:1). Then, the gonads were squashed in 45 per euchromatin does not permit identification of such cent acetic acid, the slides frozen in liquid nitrogen rearrangements. One out of four individuals and the coverslips removed by a fine razor blade. studied had an heteromorphic 9-chromosome pair formed by an acrocentric plus a metacentric chromosome (fig. 2). Bandingprocedures Freshpreparations were digested with 50-100 units of either EcoRl or AluI (Boehringer-Mannheim) DISCUSSION in 100 1sjofthe appropriate buffer and evenly spread over the slide by means of a coverslip. The Thein situ digestion of fixed chromosomes with digestions were carried out at 37°C overnight. After REs in T molitor provides an excellent tool to that, the preparations were washed with distilled identify each chromosome pair, better than C- water and stained with 2 per cent Giemsa in phos- banding or other techniques, opening the way for phate buffer (pH 6.8). Control digestions with the karyological studies in these organisms with small buffers only were also undertaken. Conventional chromosomes. We have now consistent data which C-banding (Sumner, 1972) and Giemsa staining show that the clear chromosome morphology after were also performed. digestion with EcoRI is closely related to the par- ticular structure of the satellite DNA localized in the heterochromatic regions of T molitor. We will RESULTS deal fully in the biochemical and cytological details in a separate paper (Juan et aL, in preparation). Fig.1(a) shows the karyotype of T. molitor, with In the genus Tribolium, belonging to the same meta- and submetacentric chromosomes decreas- family, Smith (1952) described heterochromatic ing somewhat in size. The X-chromosome is a blocks in all pachytene bivalents by observation medium-sized, submetacentric and the Y a dot-like of male meiosis. Petitpierre et a!. (1988) pointed chromosome. Constitutive heterochromatin out that there is a large amount of a satellite DNA revealed by C-banding occupies extensive blocks in T molitor, almost 50 per cent of the whole apparently localized in the pericentromeric regions genome. This satellite is rather rich in AT base of all chromosomes (fig. 1(b)). In five pairs the pairs (61 per cent). Therefore, chromomycin A3 (a C-bands are distal and in four pairs and the X- flurochrome which binds preferentially to GC-rich chromosome they are in the middle of the chromo- DNAs) gives rise to dull fluorescence in hetero- some. The Y-chromosome appears entirely chromatin. In any event, the level of contrast heterochromatic. between eu- and heterochromatin is not as good When the T. molitor chromosomes are treated as that obtained with EcoRl. The homogenous with A/u I, they show a C-like banding pattern with digestion (EcoRI) or null digestion (AluI) as well prominent heterochromatic blocks in each auto- as the evenness of fluorescence with CMA3 or 160 C. JUAN, J. GOSALVEZ AND E. PETITPIERRE 'I. S Ed. as1t'4a S V.0 I tt 0'\ 1 2 3 4 E'ii 1 bill 6 7 8 9 xY

Figure 2 EcoRl digested spermatogonial metaphase (a) and karyotype (b) of one individual with a chromosome heteromorphism in pair number 9 Bar 5 m.

DAPI in every centromere region, point to an compaction of the heterochromatin and the size equilocal distribution ofheterochromatin of the restriction target are important factors in throughout all the genome of T molitor such as determining the amount of removable DNA has been reported in other organisms (Schweizer (Miller et al., 1983; Mezzanotte et a!., 1983, 1985; et a!., 1987; John et a!., 1985). This homogenous Bianchi et a!., 1985). The results obtained with distribution also involves the Y-chromosome EcoRI show, at least in this species, that both which is entirely heterochromatic and entirely factors are irrelevant to RE-cleavage and sub- digested by EcoRI. This heterochromatic sequent DNA-removal. Cases of extensive homogeneity in T molitor is similar to that found cleavage of heterochromatic regions have been in mouse chromosomes (Pardue & Gall, 1970; reported in different organisms. For example, AluI Kaelbling et al., 1984; Mezzanotte & Ferrucci, in the grasshopper Pyrgomorpha conica removes 1984) and contrasts with other species where REs the centromeric DNA of all chromosomes both in have shown a remarkable heterochromatic mitosis and meiosis (Lopez-Fernandez eta!., 1989). heterogeneity (Gosálvez et a!., 1987; Lopez- In mouse Avail (GGCC) and BstNI (CCGG) Fernández et a!., 1989). reduce the staining of most of the centromeres Among a series of factors which may affect (Kaelbling et aL, 1984), although in this case, the the cleavage of fixed chromatin by a RE (Bianchi results of BstNI appears contradictory to those et aL, 1985; Mezzanotte et a!., 1985; Gosálvez et reported by Burkholder (1989). In general, four al., 1989), it has been extensively claimed that the base pair cutters are more efficient in removing ENDONUCLEASE BANDING IN TENEBRIO MOLITOR 161 large amounts of DNA, while five or six base pair ENNIS, T. i. 1974. Chromosome structure in Chilocorus (Coleop- cutters usually do not produce longitudinal tera, Coccinaelidae). I. Fluorescent and Giemsa banding differentiation or give G bands (Mezzanotte et a!., patterns. Can.J.Genet. Cytof, 16, 651. FREDLANI, M., MEZZANO1TE, R., VANNI, R., PIGNONE. D. AND 1985; Lopez—Fernandez et a!., 1989). Indeed this CREMONINI, R. 1987. The biochemical and cytological is the first case reported where a six base pair cutter characterization of Vicia lava DNA by mean of Mbo I, (EcoRI) produce a dramatic decrease in the stain- Alu I and Barn HI restriction endonucleases. Theor. app!. ing of a heterochromatic region. Genet., 75, 46-50. GOSALVEZ, J., BELLA, J. L., LOPEZ-FERNANDEZ, C. AND MEZ- The EcoRI banding in T molitor has allowed ZANOTTE, R. 1987. Correlation between constitutive us to find a hetermorphism for a chromosome pair heterochromatin and restriction enzyme resistant (pair number 9), probably caused by a perientric chromatin in Arcyptera tornosi (Orthoptera). Heredity, 59, inversion, according to the similar sizes of the 173—180. GOSALVEZ, .1., LOPEZ-FERNANDEZ, C., FERRUCCI, L. AND EcoRI resistant chromatin in both the standard MEzzANOTFE, R. 1989. DNA base sequence is not the only and the rearranged chromosome. Pericentric inver- factor for restriction endonuclease activity on metaphase sions have often been hypothesized as one of the chromosomes: evidence using isoschizomers. Cytogenet, most important structural rearrangements in Cell Gene!., 50, 142-144. HSIAO, T. H. AND HSIAO, C. 1983. Chromosornal analysis of coleopteran chromosomes, but are poorly demon- Leptinotarsa and Labidomera beetles (Coleoptera: Chry- strated because of the limited resolution of conven- somelidae). Genetica, 60, 139—150. tional staining techniques. Such rearrangements JOHN, B., KING, M., SCHWEIZER, D. AND MENDELAK. M. 1985. have been claimed in some species of Cicindelidae Equilocality of heterochromatin distribution and hetero- (Serrano, 1981), Scarabaeidae (Virkki, 1967), chromatin heterogeneity in acridoid grasshoppers. Chromosoma, 91, 185-200. Chrysomelidae (Petitpierre, 1983; Hsiao and JUAN, C. AND PETITPIERRE, E. 1989. C-banding and DNA Hsiao, 1983), among others. Moreover, it has been content of seven species of Tenebrionidae (Coleoptera). supposed that centric fissions in metacentric Genome, 32, 834-839. chromosomes were followed by pericentric inver- KAELBLING, M., MILLER, D. A. AND MILLER, 0. j.1984. Restriction enzymes banding of mouse metaphase chromo- sions or heterochromatin accretions, restoring the somes. Chromosoma, 90, 128-132. metacentric shape that prevails in coleopteran LLOYD, M. A. AND THORGAARD, G. H. 1988. Restriction chromosomes (Smith and Virkki, 1978). We think endonuclease banding of rainbow trout chromosomes. these techniques may be used to throw some light Chromosoma, 96, 171—177. on the real role that these rearrangements have in LOPEZ-FERNANDEZ, C.. GOSALVEZ, J, BELLA, J. L., DE LA TORRE, J. AND MEZZANOTTE, R. 1989. Utilización de las the divergence of closely related taxa. endonucleasas de restricción en el análisis del genoma eucarionte. In Genética. Con tribuciones de Ia Genética espano!a en homenaje a! profesor Sañudo, Fundación Acknowledgments We would like to thank Professor R. Mez- Ramón Areces. Madrid, pp. 131-149. zanotte for discussions to improve the manuscript and to Dr MEZZANOTTE, R., FERRUCCI, I. L., VANNI, R. AND BlANCH!. X. Belles for kindly providing a T molitor culture. This work V. 1983. Selective digestion of human metaphase chromo- has been supported by Projects of DGICYT no. PB87-0584- somes by Alu I restriction endonuclease. J. 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