Heredity 70 (1993) 458-465 Received 21 July 1992 Genetical Society of Great Britain Heterochromatin differentiation between two species of the genus Dociostaurus (Orthoptera: Acrididae) E. RODRIGUEZ INIGO, J. L. B ELLA & C. GARCIA DE LA VEGA* Unidad de Genética, Departamento de BiologIa, Edificio de Biologia, Facultad de Ciencias, Universidad Autónoma de Madrid, 28049 Madrid, Spain Dociostaurusjagoi and Dociostaurus genei are two acridoid grasshoppers which show large differ- ences in heterochromatin content. The use of several banding techniques, including fluorochrome staining and restriction endonuclease digestion, provides further information on the characteristics of the constitutive heterochromatin regions revealed with the C-banding method. Thus both species show GC-enriched bands accompanying active nucleolar organizers which differ in location. Because of its bright response to DAPI and CMA3, the centromeric heterochromatin seems to have a bipartite nature in terms of DNA composition in D. genei, while it shows dull staining with both fluorochromes in D. jagoi. However, two endonucleases, MboI and Sau3A, extensively digest these regions in both species. The supernumerary heterochromatic segments present only in D. genei seem to be AT-rich and are extensively digested with Alul. These results reveal heterogeneity in the distinct C-banded regions which, in turn, are equilocally distributed in both chromosome complements. Keywords:chromosomebanding, Dociostaurus, insect cytogenetics, supernumary hetero- chromatin. Introduction Soltani (1978) described sharp differences in male genitalia and number of stridulatory pegs. In this paper Constitutiveheterochromatin, usually revealed by we report chromosome differentiation between both C-banding techniques, is a common component of species, mainly involving the heterochromatin content. eukaryotic genomes that presents large variation in Moreover we have assayed several banding techniques content and distribution both between and within to further characterize the different heterochromatic species (John, 1988). Other banding techniques, such regions. Results obtained support two common as fluorochrome staining or digestion with restriction features of heterochromatin: heterogeneity and equi- enzymes, have revealed that heterochromatin may locality. show important differences in composition (hetero- geneity) and it is commonly restricted to certain chromosome regions within a given complement Materialand methods (equilocality). In Acridoid grasshoppers, general surveys of Iberian (Santos et al., 1983) and Australian Adultmales of D. jagoi and D. genei collected from (King & John, 1980) grasshoppers demonstrated natural populations in central Spain were dissected and dramatic changes in heterochromatin content and the testes fixed in ethanol:acetic acid (3:1). To carry distribution even between closely related species. D. out the different banding techniques squash prepara- jagoi and D. genei are two species widely distributed in tions were performed in a drop of acetic acid 45 per drylands of south-western Europe, North Africa and cent, the coverslips were removed after freezing in the Middle East. They are commonly found in liquid nitrogen and the slides were then air-dried. sympatry. On morphological grounds these species C-banding was performed according to Lopez- were considered as a single one up to 1978, when Fernández & Gosálvez (1981). The silver impregnation method was that developed by Rufas & Gosálvez *Correspondence. (1982). Fluorochrome banding with 4,6 diamidin 2 HETEROCHROMATIN DIFFERENTIATION 459 phenylindole (DAPI), distamycin A (DA) and chromo- mycin A3 (CMA3) was obtained according to Ag-NORs Schweizer (1980). In situ digestion with restriction Throughoutthe first meiotic prophase two nucleoli are enzymes (REs) was carried out according to observed in both species. However their chromosome Mezzanotte et al. (1983). location is clearly distinct. In D. jagoi the nucleolar remnants appear associated to an interstitial region of the Li bivalent, and the centromere region of a short Results bivalent (Fig. ia), while in D. genei nucleoli are asso- Thechromosome complement of both species is com- ciated to the centromeric region of the two short biva- posed of 23 (males) and 24 (females) acro-telocentric lents (Fig. ib). chromosomes. According to their size they can be grouped in three long (L1—L3), five medium (M4—M8) and three short (S9—Sii) pairs. The X chromosome, C-bandingpatterns single in males (XO) and double in females (XX), is the Inboth species every chromosome shows pericentro- 4th in decreasing size (Garcia de la Vega, 1984). meric C-bands. However, these bands are larger in D. p - C eta 4 '' a -. , a' !t 0) I2' U 'A ¶ fret e isa w a. 0• xl... RI 41 St r S Fig. I (a) and (b) Silver-stained diplotene cells of D. jagoi (a) and D. genei (b) showing two active NORs in both cases but with different location (arrows). (c) and (d) C-banded first meiotic metaphases of D. jagoi (c) and D. genei (d). Every chromosome has pericentromeric bands although those of D. genei are bigger than those of D. jagoi. In this species the Li bivalent has an inter- stitial band (arrow). Moreover most of the autosomes of D. genei have distal supernumerary segments (arrows). X: sex chromo- some. 460 E. RODRIGUEZ IFJIGO ETAL. genei than those found in D.jagoi(cf. Fig. ic and d). reveals bright bands in the centromere regions of D. Moreover seven chromosome pairs (M5—S11) of D. jagoi(Fig. 2a and c), whereas the centromeric C-bands geneimay show large distal heterochromatic blocks of D.geneishow complex composition (cf. Figs id, 2b (Fig. id). At least five of the pairs show polymorphism and d). Thus the proximal region appears bright with for the presence of these supernumerary segments. The DAPI (Fig. 2b), while DA-CMA3 reveals tiny juxta- regions where NORs have been detected also show centromeric bands below those differentially stained accompanying C-bands. This is particularly obvious in with DAPI in most of the chromosomes (Fig. 2d). The the case of the Li pair of D.jagoiwhere an interstitial supernumerary segments present in D.geneiare band is always present (Fig. ic). The C-bands asso- enriched in AT regions, as revealed by the bright bands ciated with the other active NORs of both species are obtained with DAPI. These regions, in turn, appear closely located to the centromeric bands of the corre- negative with DA-CMA3 (cf. Fig. 2b and d). Finally, the sponding chromosomes. heterochromatin associated with active NORs appears bright with DA-CMA3 in both species (Fig. 2c and d). Fluorochrome banding patterns Restrictionendonuclease banding patterns Centromereheterochromatin of both species can be differentiated when fluorochromes specific for AT Chromosomesof both species were digested in situ (DAPI) or GC (Chromomycin A3) DNA base pair with several REs (Tables 1 and 2). Most of the enzymes enriched regions are assayed. Neither fluorochrome employed reproduce C-like banding patterns; that is, Fig. 2 Fluorochrome banding. (a) and (b) First metaphases of D.jagoi(a) and D.genei(b) stained with DAP1. Both the centro- meres and the distal segments of D.genei(b) appear bright. In D.jagoi(a) C-banded regions show dull fluorescence. Second metaphases of D.jagoi(c) and D.genei(d) stained with DA-CMA3. In D.jagoitwo half-bivalents show bright bands that corre- spond with the active NORs detected with silver staining. Some chromosomes of D.geneishow paracentromeric positive bands. In this species neither the centromeric regions nor the distal blocks show bright fluorescence. HETEROCHROMATIN DIFFERENTIATION 461 Table 1 Characterization of the heterochromatic regions of Table 2 Characterization of the heterochromatic regions of D.jagoi D. genei NORs CEN CEN C-banding + + NORs PROX JUXTA SS DAPI - 0 C-banding + + + + DA-CMA3 + 0 DAPI — + 0 + REs DA-CMA3 + 0 + 0 + + A/uI (AG/CT) REs BamHI(G/GATCC) + + Dral (TTT/AAA) + + AluI(AG/CT) + + + - EcoRl(G/AATTC) + + BamHI (G/GATCC) + + + + Haelll (GG/CC) — + Dra! (TTT/AAA) + + + HpaII(C/CGG) — + EcoRl(G/AATTC) + + + Hinfl(G/ANTC) + + HaelIl(GG/CC) — + — + MboI(/GATC) 0 — HpaII(C/CGG) — + + Sau3A (/GATC) 0 — Hinfl(G/ANTC) + + + + MboI (/GATC) 0 — — + NORs: nucleolar organizer regions; CEN: pericentromeric Sau3A (/GATC) 0 — — + regions. +:positive staining; —:negative staining; 0: dull PvuII (CAG/CTG) + + + + staining. NORs: nucleolar organizer regions; CEN: pericentromeric regions. PROX: proximal regions; JUXTA: juxtacentromeric regions: SS: supernumerary segments. chromosome arms are extensively digested except +: staining; 0: dull staining. where constitutive heterochromatin regions occur. positive staining; —:negative However, some of them have provided clear-cut differ- ences between the heterochromatic regions. Thus HaeIII and HpaII, two enzymes containing only G and assume the concerted evolution of similar repetitive C in their targets, digest extensively the G/C enriched DNA sequences in equivalent chromosome arms at regions of both complements, i.e. the heterochromatin similar positions (Schweizer et a!., 1983; Schweizer & associated with NORs as well as the juxtacentromeric Loidl, 1987). A comparative study of heterochromatin bands of several chromosomes in D. genei (Fig. 3a distribution and heterogeneity in 10 species of acridoid andb). grasshoppers showed the strong tendency within a Te isoschyzomeres MboI and Sau3A digest the given complement for the accumulation of hetero- centromeric regions of both species while, to a large chromatin