Heredity 56 (1986) 237—241 The Genetical Society of Great Britain Received 22 July 1985 Extra nucleolar activity associated with presence of a supernumerary chromosome segment in the grasshopper Oedipoda fuscocincta J. P. M. Camacho, J. Navas-Castillo Departamento de Genética, Facultad de Ciencias, and J. Cabrero Universidad de Granada, 18071 Granada, Spain. The standard males of Oedipoda fuscocincta show a single active nucleolus organiser region (NOR) located in the secondary constriction of the M9 chromosomes. Nine males from three different populations proved to be heterozygous for a heterochromatic supernumerary segment which is distally located on the S10 chromosome. This also showed NOR activity. All available data seem to support the hypothesis that this extra segment on the S10 chromosome has arisen by translocation of part of the M9 chromosome, specifically that located between the centromere and the secondary constriction. INTRODUCTION However, in this paper we report a supernu- merary chromosome segment containing an active Oneform of chromosomal polymorphism com- NOR, which may serve as evidence in favour of monly identified in acridid grasshoppers is that the origin of this extra segment by means of translo- caused by the presence of extra chromatin seg- cation. ments attached one or more chromosomes of the standard complement. That is to say they are super- numerary chromosome segments. The majority of MATERIALSAND METHODS observed extra segments are heterochromatic in nature, although two cases of euchromatic ones Forty-sevenadult males of the grasshopper have been recorded in the gomphocerine grasshop- Oedipoda fuscocincta Saussure were caught at five pers namely, Omocestus bolivari (Camacho et a!., localities in the South of the Iberian Peninsula, 1984) and Chorthippus binotatus (Cabrero, 1985). specifically five males at Dornajo (Sierra Nevada, Those extra segments which are heterochromatic, Granada), two males at Las Sabinas (Sierra as can be deduced from their heteropycnosis dur- Nevada, Granada), three males at Sierra de Pizarra ing first meiotic prophase, may show four types of (Málaga), two males at Sierra de Cázulas response to C-banding (Camacho et a!., 1984), (Granada) and 35 males at Alcalá la Real (Jaén). which indicates their heterogeneous nature. A mat- Testes were fixed in acetic ethanol (1:3) and ter which has long caused controversy and still subsequently were studied cytologically by the remains unresolved is the origin of supernumerary C-banding technique described in Camacho et a!. segments. White has (1954; 1973) proposed two (1984) and the silver impregnation technique of mechanisms by which extra segments may arise, Rufas et al. (1982). firstly by translocation of chromosome material from the standard complement or else from a B chromosome, and secondly by duplication and RESULTSAND DISCUSSION heterochromatinisation of some part of the genome. The second hypothesis has been accepted Likeother species belonging to the subfamily by most authors (Shaw, 1970; 1971; John 1973; Oedipodinae (Cabrero and Camacho, 1982; Hewitt, 1979; Camacho and Cabrero, 1982; Camacho and Cabrero, 1983), Oedipoda fus- Camacho et a!., 1984) and indeed many arguments cocincta has a chromosome complement consisting have been put forward against the translocation of 2n =22+XO/XXtelocentric chromosomes. hypothesis (Hewitt, 1979). The autosomes can be classified into three size 238 J. P. M. CAMACHO, J. NAVAS-CASTILLO AND J. CABRERO groups: three long (L1-L3), six medium (M4-M9) secondary constrictions behave like "elastic con- and two short (S10 and S11), the X chromosome strictions" (White, 1957; John and Naylor, 1961; being the fourth element in size (Camacho, 1980). Mesa and de Mesa, 1967; John, 1976) in some cells The M9 behaves like a megameric bivalent during at first meiotic prophase (fig. 1(b)—(d)). first meiotic prophase, appearing positively In three out of the five populations sampled heteropycnotic like the X chromosome (fig. 1(a)). (Dornajo, Sierra de Cázulas and Alcalá la Real), The M9 bivalent possesses a secondary constriction we detected the presence of a heterochromatic near the centromere, just like that in 0. coerule- extra segment distally located in the S10 chromo- scens, 0. charpentieri and species of the related some. This extra segment was carried by a total of genus Sphingonotus (Camacho, 1980). These nine heterozygous males, specifically two from F .;zs:1 C?;> esx C:- H? 'cc a )ç Mg 4 x's'. 4. •t,%Y. \.0 L Figure 1 "Elastic constrictions" (arrows in (b)—(d)) in Oedipoda coerulescens. (a) Normal cell. (b) Cell showing the secondary constriction of one M9 chromosome behaving like an "elastic constriction". (c) and (d) Cells showing both secondary constrictions in the M9 bivalent behaving like "elastic constrictions". EXTRA NUCLEOLAR ACTIVITY ASSOCIATED WITH SUPERNUMERARY CHROMOSOME 239 Dornajo, one from Sierra de Cázulas and six from in which a nucleolus appears joined to each active Alcalá la Real. In previous papers we reported the NOR so that bivalents carrying them are easily existenceofthissupernumerary segment recognisable. (Camacho and Cabrero, 1982) and its positive In standard males, the silver impregnation tech- reaction to C-banding (Camacho et a!., 1984) (see nique reveals the presence of a single nucleolus at also fig. 2(a)). In the present investigation, we have zygotene and which in diplotene cells appears analysed the activity of nucleolus organiser regions joined to the secondary constrictions of the M9 (NORs) in standard and S10-segmented males by bivalent (fig. 2(b)). Hence, this species possesses means of the silver impregnation technique, which a single active NOR located in the secondary con- selectively stains the transcriptionally active NORs striction of the megameric bivalent. However, in (Miller et al., 1976a, b). In grasshoppers, active males heterozygous for the supernumerary seg- NORs may only be demonstrated in the primary ment on the S10 chromosome, silver impregnation spermatocytes at pachytene and diplotene stages, reveals the pesence of two nucleoli in zygoten p .. .1 ¼ ¶ 4 is 4 v' S10ft V4q x " ' ,1 I • a S a, —. S I' b hr t) St{AflUc i at t o e• •' a /4. • b •4 4 MPV'SjP jr Figure 2 Nucleolar activity in Oedipodafuscocincta.(a)Metaphase II showing the extra segment darkly C-banded (arrow). This cell is derived from a primary spermatocyte in which the S10 heteromorphic bivalent divided equationally. (b) Silver stained diplotene cell from a standard male. Note the presence of nucleoli (nu) joined to the active NORs located in the secondary constrictions of the megameric M9 bivalent, and the absence of nucleolar activity in the basic S10 bivalent. (c) and (d) Silver stained cells from males heterozygous for the extra segment in the S10 bivalent. Note nucleolar activity in the M9 bivalent and also in the supernumerary segment of the S10 bivalent. Arrows point to the extra segments, nu =nucleolus.(e) Silver stained partial diplotene cell showing NOR activity in the M9 and the heteromorphic S10 bivalent. Note the similar size of the extra segment (arrow) and the zone of M9 lying between centromere and the secondary constriction. Note the proximal location of the single chiasma in the hetromorphic S10 bivalents (c)—(e) but the distal location in the basic ones (b). 240 J. P. M. CAMACHO, J. NAVAS-CASTILLO AND J. CABRERO cells which in diplotene cells appear joined to the polysomic males of grasshoppers (Hewitt and active NORs located in the secondary constrictions John, 1968; 1970; Fox et a!., 1974; Camacho et a!., of the M9 bivalent and, additionally, in the zone 1981; Viseras and Camacho, 1984). However, it is by which the extra segment is joined to the S10 clear that this heterochromatinisation has not chromosome (fig. 2(c)-(e)). This additional NOR affected the activity of the NOR in its new location in the extra segment of the S10 was present in the in the S10 chromosome. nine heterozygous males analysed from three different populations. However, thisextra nucleolar activity was never observed in the S10 Acknowledgements We would like to thank Ms Merce Romero chromosomes lacking the supernumerary segment for her aid in collecting the grasshoppers. or in the S10 chromosomes of standard males. Thus it may be concluded that the extra NOR is con- tained in the supernumerary segment. The location REFERENCES of ribosomal RNA genes in supernumerary chromosome material has only been reported for BABCOCK,E. .1947. Thegenus Crepis, part two. Univ. Ca!f the accessory chromosomes of several plant Bot., 22. BArrAGLIA,E. 1964. Un secondo caso de B-cromosomi (2n = species: Crepis syriaca (Cameron, 1934), C.foetida 14+6—8B) in Scilla autumnalis L. (Liliaceae)proveniente vulgaris (Babcock, 1947), Alopecurus pratensis dalla Palestina. Caryo!ogia, 17,65—76. (Bosemark, 1957), Pennisetum typhoides (Powell BOSEMARK, N. 0. 1957. Further studies on accessory chromo- and Burton, 1966), Gibasis linearis (Brandham and somes in grasses. Hereditas, 43, 236-297. BRANDHAM, D. E. AND BHAYFARAI, C. 1977. The effect of Bhattarai, 1977), Calycadenia (Carr and Carr, B-chromosome number of chiasma frequency within and 1982), Allium flavum (Loidi, 1982) and A. between individuals of Gibasis linearis (Commelinaceae), sphaerocephalon (Guillén and Ruiz Rejón, 1984). Chromosoma, 64, 343-348. However, their presence in supernumerary CABRERO, .t. 1985. Estudios citogenéticos en saltamontes de Ia chromosome segments has now been detected for subfamilia Gomphocerinae: heterocromatina,reor- denaciones