Introduction

Eukaryote genomes are structured in a number of standard chromosomes (A) and, in many species, a variable number of supernumerary, B, chromosomes that behave as genome parasites (Camacho et al, 2000). B chromosomes have been reported in more than 1300 plant and 500 animal species (Jones and Rees, 1982; Jones and Puertas, 1993; Jones, 1995), and Beukeboom (1994) estimated that about 15% of described living beings harbour polymorphisms for B chromosomes. The nature of these chromosomes is heterogeneous, although they are predominantly heterochromatic and show a high variety of morphologies, chromosomal banding patterns and molecular compositions (Henriques-Gil et al, 1984; Green et al, 1987; Green, 1988; López-León et al, 1993; Bakkali et al, 1999; Cabrero et al, 1999). In Orthoptera, which is the animal group where B chromosome polymorphisms have been more frequently reported (Jones and Rees, 1982), B chromosomes are commonly telocentric, but there are also some iso-B chromosomes originated through centromere misdivision and chromatid nondisjunction, or by centric fusion between two B chromosomes (see López-León et al, 1993).

The karyotype of the grasshopper Eyprepocnemis plorans is composed of 2n=22+X0 in males and 2n=22+XX in females. The autosomes may be classified into three size groups: Long (L₁–L₂), Medium (M₃–M₈) and Small (S₉–S₁₁), the X chromosome size being intermediate between those of L₂ and M₃. Previous studies have shown that almost all E. plorans populations analysed from the Mediterranean region and the Caucasus show the polymorphism for B chromosomes (Camacho et al, 1980; Henriques-Gil et al, 1984; López-Fernández et al, 1992; López-León et al, 1993; Cabrero et al, 1997; Bugrov et al, 1999). This polymorphism has revealed the existence of an arms race between A and B chromosomes leading the system through successive stages with B chromosomes passing from parasitic to near-neutral (Camacho et al, 1997) and vice versa, thus facilitating B-polymorphism regeneration (Zurita et al, 1998). A crucial requisite for this regeneration is a high mutability of B chromosomes, a property that has been observed in Spanish B chromosomes (López-León et al, 1993).

All North African E. plorans populations hitherto analysed showed the presence of the B chromosome polymorphism. Henriques-Gil and Arana (1990) reported the presence, in Melilla, of a predominant B variant that they named B₁₆. Later, Bakkali et al (1999) analysed nine Moroccan populations where the predominant B variant was similar, in morphology and C banding pattern, to the Spanish B₁ described by Henriques-Gil et al (1984). The present work analyses the mutability of B chromosomes in nine Moroccan populations of the grasshopper E. plorans, and estimate B chromosome mutation rate in controlled crosses between B-carrying females and 0B males.

Materials and methods

Adult specimens of the grasshopper E. plorans were collected for 3 consecutive years (1995–1997) at nine natural populations in North-Western Morocco: Smir, Asilah, Larache, Ain l'abid, Tatouft, Frain, SO.DE.A., Mechra Bel Ksiri (to which we call Mechra) and Rabat (see map locations in Bakkali et al, 1999). Most animals were fixed for cytological analysis in Morocco to avoid death risks during transport to Spain. Some specimens caught at Smir, SO.DE.A. and Mechra were taken alive to the laboratory to perform controlled crosses between B-carrying females and 0B males. Live specimens were bred in cages similar to those described in Clemente et al (1985), in a culture room with 10:14 light:dark photoperiod, and 28°C. Culture conditions were similar to those described in Herrera et al (1996). To facilitate controlled crosses, we performed an in vivo analysis of B chromosome presence in males, which were vivisected for the extraction of some testis follicles by means of a little cut in the median dorsal part of the abdomen. Owing to mortality risks, in vivo study of females was not carried out. For this reason, and to increase the likelihood that females were B-carriers, we firstly obtained a stock of animals with increased B frequency (the progeny of B-carrying males) (see details in Bakkali, 2001; Bakkali et al, 2002). Female virginity in this stock was preserved, for controlled crosses, by separating males and females in the preadult nymph stage. Egg pods were collected from females used in the controlled crosses.

For cytological analysis of adults, testes were fixed in 1:3 acetic acid-ethanol and stored at 4°C, and females were injected with 0.2 ml of 0.05% colchicine in insect saline solution 6 h prior to anaesthesia, dissection and extraction of ovaries, which were fixed and stored like testes. For the cytological analysis of embryos, eggs were dissected in insect saline solution, after 11 days of incubation at 28°C, and embryos were immersed in 1 ml of 0.05% colchicine in insect saline solution for 45 min. Hypotonic treatment was performed by adding 1 ml of distilled water for 10 min. Finally, embryos were fixed in 1:3 acetic acid–ethanol.

Cytological preparations from adult materials were carried out by squashing two testis follicles (or two ovarioles) in acetic orcein for B presence detection, or in 50% acetic acid (and then removing the coverslip after freezing in liquid nitrogen) to perform C banding. Embryo preparations were made by a method similar to that used by Meredith (1969) for the study of mammal meiosis (see details in Camacho et al, 1991). C banding was performed by the technique described in Camacho et al (1984).

To name the different B chromosome variants found in Morocco, and avoid confusion with the names used in Spain (B₁, B₂, etc), we have added an M to the subscript indicating order of finding (B_M2, B_M3, etc). Excepting B₁, which was essentially similar to the Spanish one (see Cabrero et al, 1999; Bakkali et al, 2002), coincidence in the classificatory number between Moroccan and Spanish B chromosomes does not necessarily reflect similarity between them. Table 1 shows the equivalence (wherever possible) between Moroccan and Spanish B variants.

Table 1 - Description and possible origin of the 15 B chromosome variants found in the six Eyprepocnemis plorans Moroccan populations.

Full table

Mutation rate was calculated from the de novo origin of B variants in controlled crosses. For this purpose, we scored the number of embryos carrying the maternal B chromosome as well as those carrying a different B variant. The detectable mutation rate per B chromosome (u_B) depends on the total number of embryos analysed. It is calculated as the reciprocal of the product between the total number of embryos analysed (N) and the mean number of Bs found in the sample (M_B): u_B=1/(N M_B). The mutation rate of a B chromosome is u_B times the number of embryos carrying mutations involving this B chromosome.

Results

In addition to the principal B chromosome, B₁, the only one present at a relatively high frequency in all Moroccan populations analysed by us (see Bakkali et al, 1999), we found 15 other minority B variants that were distinguishable on the basis of their morphology and C banding pattern (Figure 1 and Table 1). Eight variants were found in adults caught in the wild, four appeared in adults reared in the laboratory and seven were found in embryo progeny of controlled crosses. As Table 2 shows, some of these variants were found in more than one kind of material. All B variants showed very low frequency, and seven of them (shown in Table 3) were found in embryos whose mother carried a different B variant. The only conceivable mode of origin for these variants is through mutation of the maternal variant, which can be considered the ancestral one.

Figure 1.

Morphology and C banding pattern of the 16 B chromosomes found in the nine Moroccan populations studied in this work. B₁ is the principal B chromosome found in all of these populations, the remaining are minority B chromosomes found in some adults from the natural populations or in embryos and adults obtained in our laboratory (see Table 2).

Full figure and legend (46K)

Table 2 - Frequency of 10 B variants found in adult specimens from Moroccan populations of Eyprepocnemis plorans.

Full table

Table 3 - Frequency of B chromosome variants arisen in controlled crosses performed in the laboratory between a 0B male and a B-carrying female.

Full table

Several individuals were additionally found to carry chromosomal translocations involving B and A chromosomes. An adult male carried a reciprocal translocation between the B_M8 variant and a medium-sized autosome (M₄) (see a detailed study in Bakkali et al, 2003), two embryos carried B–A Robertsonian translocations (with the simultaneous observation of a mini-chromosome) and another embryo carried a B–A centric fusion (with no mini-chromosome) (Figure 2). One of the Robertsonian translocations occurred between B₁ and the M₅ autosome and the other involved B_M3 and the smallest autosome (S₁₁). The centric fusion occurred between B_M7 and the third autosome (M₃).

Figure 2.

C banding of the chromosomes produced through A–B chromosome translocations. M₄–B_M8 and B_M8–M₄ were found in and adult male caught at the population of Smir, the remaining had arisen in controlled crosses performed in the laboratory. The dotted line joins the centromeres of all chromosomes. The vertical lines indicate the A chromosome region in each mutant chromosome.

Full figure and legend (43K)

The average mutation rate for all types of maternal B chromosomes analysed in controlled crosses was 0.73% (Table 4), but there were conspicuous differences between variants, with B_M7, an isochromosome, being the most unstable B chromosome (9.64%) giving frequently rise to the B_M12 variant through centromere misdivision (see Tables 1 and 3) and being involved in a B–A translocation (see Figure 2). In total, 20 mutations were found involving B chromosomes, 20% of which were translocations with A chromosomes, 20% were translocations between B chromosomes, and the remaining 60% were mutations involving a single B chromosome. Most single B mutations were centromere misdivisions with or without later chromatid nondisjunction to give rise, respectively, to iso-B-chromosomes (eg B₁ becoming B_M4, or B_M3 becoming B_M13) or telocentric Bs (eg B_M7 becoming B_M12) (see Tables 3 and 4). The only exception was an embryo from the Smir population carrying B_M11 presumably arisen from the maternal B, B_M2, through deletion of most rDNA (see Figure 1).

Table 4 - Mutation rate of B chromosomes in populations from Morocco, as deduced from the analysis of embryo progeny raised in controlled crosses between a 0B male and a B-carrying female.

Full table

Discussion

On the basis of morphology and C banding pattern, more than 50 B variants have been described in natural populations of E. plorans (Henriques-Gil et al, 1984; López-León et al, 1993). The B₁ chromosome was considered the original B in Spanish populations (Henriques-Gil et al, 1984). Given its predominance in all nine Moroccan populations analysed, we also considered B₁ as the original B in Moroccan populations (Bakkali et al, 1999). In Melilla, however, the most frequent B chromosome variant (B₁₆), reported by Henriques-Gil and Arana (1990), was different from B₁. Since this population is separated from the Moroccan populations analysed in Bakkali et al (1999) by the Rif mountains, and from Spanish populations by the Mediterranean sea, it is likely that the B chromosome polymorphism has followed an independent evolutionary pathway in Melilla, although this subject requires further study.

Although B₁ is well established in all nine populations analysed, our present results show that new B chromosome variants are arising through different mutations, at least in six out of these populations. Bearing in mind that only B chromosomes showing drive, or being beneficial for the host, are expected to become frequent in natural populations (Camacho et al, 1997), it is likely that most of the rare B variants found in Morocco, at very low frequency, are either of very recent origin or else neutral or deleterious (it is reasonable to assume that it is unlikely that a beneficial B variant could appear by mutation of a parasitic B). Since B chromosomes lack a regular meiotic behaviour (they do not go in pairs that pair and segregate during meiosis, as A chromosomes do), a neutral B variant cannot reach fixation by drift, but only be eliminated. In this scenario, the observation of many B variants at low frequency is explained by high mutation rate (0.73% on average) compensating loss by drift, although we cannot rule out that any of these rare variants could prosper through drive.

The most frequently observed B mutation was centromere misdivision with chromatid nondisjunction giving rise to iso-B-chromosomes. Translocations with A or B chromosomes were also frequent, most of them implying centromere breakpoints, but only one deletion of rDNA was observed. The complete range of B variants (see Figure 1 and Table 1) suggests that other mutations, such as duplications, inversions and centric fusions, can also affect B chromosomes.

A very similar situation has been reported in Spanish populations, where the de novo formation of 14 B chromosome variants was found in the progeny of controlled crosses and gravid females, the formation of iso-B-chromosomes being also the most frequently observed mutation, but tandem and centric fusions, pericentric inversions and deletions were also observed (López-León et al, 1993). Since these 14 B variants were found among a total of 3012 embryos whose average B frequency was 0.787, the detectable mutation rate per B chromosome was 0.042 in that sample, and the average mutation rate for B chromosomes (excluding B–A translocations that were not reported) was 0.59%, a figure being remarkably similar to the observed in Morocco: 0.437+0.146=0.583% (see Table 4). This similarity suggests that B chromosome mutability depends more on B intrinsic factors than on A-chromosome or environmental factors.

These possible factors might include centromere instability, breakage of repetitive DNA (rDNA or satDNA) and transposable elements. The prevalence of centromere misdivision among the observed chromosome mutations suggests that centromere is the most unstable region in the E. plorans B chromosomes. This instability might derive from its heterochromatic nature and/or the presence of repetitive DNA in its vicinity, that is, the 180 bp sat DNA (López-León et al, 1994, 1995). This would be consistent with the finding that centromere is a repeated structure that can be split into smaller fragments that are still able to work properly (Zinkowski et al, 1991). In maize, the size of B-chromosome centromeres, produced by misdivision, is strongly correlated to meiotic transmission and the presence of B-specific DNA repeats in the centromere (Kaszás and Birchler, 1996, 1998). However, it is also possible that centromere propensity to breakage is associated to the presence of mobile elements, which have been shown to be frequent in B chromosomes, for example, in Nasonia vitripennis (McAllister, 1995). Preliminary data, however, suggest that mobile elements such as Gypsy and Mariner are absent from repetitive DNA and centromeric regions of E. plorans B chromosomes (López-León et al, unpublished), although it does not rule out the possible implication of other unknown elements associated to B centromere instability. In fact, Gypsy and Mariner are located between the satDNA blocks in Spanish B₁, B₂ and B₂₄ and, remarkably, the Moroccan B_M7 arose by breakage at this level presumably followed by neocentric activity, which may be associated to the presence of repetitive DNA (Alfenito and Birchler, 1993; Manzanero et al, 2002).

The high mutability of E. plorans B chromosomes is tolerated because of their dispensable nature, but it promotes their evolution by increasing the likelihood of generating new parasitic variants and thus constitutes one of the main B-chromosome weapons in their evolutionary arms race with A chromosomes (Camacho et al, 1997). Our present work suggests that this high mutability is most likely due to B-chromosome intrinsic factors, the nature of which will be the subject for future research.

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Acknowledgements

M Bakkali thank the Universities of Abdelmalek Essaadi (Morocco) and Granada (Spain), the 'Agencia Española De Cooperación Internacional, Instituto de Cooperación con el Mundo Arabe, Mediterráneo y Paises en Vias de Desarrollo' (Spain) and the 'Ministère de l'Enseignement Supérieur, de la Formation des Cadres et de la Recherche Scientifique' (Morocco) for conceding studentships. We thank Mr Soulaïmane Bakkali for his help in capturing specimens. This study was supported by grants from the Spanish Ministerio de Ciencia y Tecnología (BOS2000-1521) and Plan Andaluz de Investigación, Grupo no. CVI-165.