Distributive pairing in the grasshopper Chorthippus binotatus

M. D. LOPEZ-LEON, J . CABRERO, AND J. P. M. CAMACHO Departamento de Biologlh Animal, Ecologlh y Genetica, Facultad de Ciencias, Universidad de Granada,

18071 Granada, Spain

Corresponding Editor: J . Sybenga

Received July 25, 1990

Accepted September 24, 1990

LOPEZ-LEON, M. D., CABRERO, J., and CAMACHO, J. P. M. 1991. Distributive pairing in the grasshopper Chorthippus binotatus. Genome, 34: 139- 143.

Six males of the grasshopper Chorthippus binotatus were mosaic for the presence of extra (E) chromosomes in the germ line, but lacked them in the somatic cells of gastric caeca. E chromosomes were very similar to the X chromosome in size and meiotic properties (heteropycnosis and autopairing). X and E chromosomes associated frequently at diplotene, but the associations never persisted until metaphase I, which indicated that they were not chiasmate. When one E was present, X and E univalents segregated preferentially to opposite poles. In cells with two E, they formed a bivalent in almost all cells, and decreased the frequency of X-E associations by 20%. These cells showed a high frequency of nondisjunction between the two E chromosomes, such that they segregated independently despite the high persis- tence of their association at metaphase I. These results are interpreted and discussed in the light of the distributive pairing model.

Key words: distributive pairing, X chromosome, E chromosome, Chorthippus, male meiosis.

LOPEZ-LEON, M. D., CABRERO, J., et CAMACHO, J. P. M. 1991. Distributive pairing in the grasshopper Chorthippus binotatus. Genome, 34 : 139-143.

Chez six miiles de la sauterelle Chorthippus binotatus, les cellules de lignee germinale ont presente une mosai'que de chromosomes E surnumeraires, lesquels etaient absents dans les cellules somatiques du caecum gastrique. Ces chro- mosomes E avaient beaucoup de ressemblance avec les chromosomes X, tant par leurs dimensions que leurs proprietes meiotiques, dont l'heteropycnose et l'auto-appariement. Les chromosomes X et E se sont frequemment associes au stade diplotene mais leurs associations ne persistaient pas jusqu'a la metaphase I, indice qu'elles n'etaient pas de nature chiasmatique. Lorsqu'un seul E etait present, les univalents X et E ont segrege aux p6les opposes de faqon preferen- tielle. Lorsque deux E etaient presents, ils ont forme un bivalent chez presque toutes les cellules; la frequence des asso- ciations X-E a alors ete reduite d'environ 20%. Dans ces cellules, la frequence de non-disjonction entre deux chromo- somes E a ete elevee et, malgre la grande persistance de leur association a la metaphase I, ils ont segrege independamment. Ces resultats sont interpretes et discutes a la lumiere d'un modele d'appariement distributif.

Mots cles : appariement distributif, chromosomes X, chromosomes E, Chorthippus, meiose miile. [Traduit par la redaction]

Introduction In the present paper we report a case of nonrandom "Distributive pairing refers to that phase of the meiotic segregation between the X chromosome and a polysomic

cycle during which chromosomes that had previously failed extra chromosome (E) in the grasshopper Chorthippus to undergo exchange pairing may enter into segregational binotatus. The particularities of this nonrandom segrega- associations with other non-crossover chromosomes, homol- tion closely fit those of the distributive pairing model. ogous or nonhomologous" (Grell 1962, 1976). When more than two noncrossover chromosomes are present, distri- butive pairing is competitive and preferential. Preferences are dependent on size and independent of homology. Size specificity generally favors pairing of homologues over nonhomologues. Since distributively paired homologues, like homologues with a chiasmate association, segregate regularly, the genetic identification of distributive pairing is usually based on the analysis of the abnormal patterns produced by the distributive pairing between nonhomol- ogous chromosomes.

Male meiosis in grasshoppers is an excellent material to test nonhomologous pairing and segregation between several types of additional heterochromatic chromosomes (B chro- mosomes or polysomy) and the single X chromosome. Meiotic association between the X univalent and additional chromosomes is frequent and is based on heteropycnotic affinity, but not on chiasma formation. These associations

Materials and methods Seventy-eight adult males, 25 adult females, and 38 nymph

females of Chorthippus binotatus were caught at El Navazo in the Sierra Nevada mountains (Granada, Spain). Testes were extracted from the males through a small dorsal cut in the third abdominal segment. The males were subsequently injected with 0.05'70 colchicine in insect saline solution, and 6 h later gastric caeca were fixed in acetic acid - ethanol (1:3). Females were injected with 0.05% colchicine in insect saline solution for 6 h prior to fixation of the ovarioles and gastric caeca. All materials were analyzed cyto- logically by the C-banding technique described by Camacho et al. (1984).

Results Standard individuals of Ch. binotatus have 2n = 16 +

XO/XX subtelocentric chromosomes, the autosomes being classified by size into three long (Ll-L3), four medium (M4-M7), and one short (S8), the X chromosome being

have been presumed to lead, in several cases, to nonrandom shorter than L3 but longer than M 4 (Cabrero and Camacho segregation between the X and B chromosomes (Henderson 1986). 1961; Jackson and Cheung 1967; Fontana and Vickery 1973; Of the 78 males examined, 6 carried additional extra (E) Bidau 1986). chromosomes in the testes but lacked them in gastric caeca Pr~nled in Canada / lmprime au Canada

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140 GENOME, VOL. 34, 1991

TABLE 1. Number (%) of follicles with 0 , 1, 2, and 3 extra chromosomes (E) in six males of Chorthippus binotatus

Specimen No. 2n = 17 + OE 2n = 17 + 1E 2n = 17 + 2E 2n = 17 + 3E Total - -

3 1 (4.55) 7 (31.82) 12 (54.55) 2 (9.09) 22 10 8 (21.05) 9 (23.68) 19 (50.00) 2 (5.26) 38 20 2 (12.50) 0 14 (87.50) 0 16 26 3 (12.50) 9 (37.50) 1 1 (45.83) 1 (4.17) 24

Cross 4* 2 (16.67) 6 (60.00) 4 (33.33) 0 12 Cross 13* 3 (14.29) 6 (28.57) 10 (47.62) 2 (9.52) 2 1

Total 19 (14.29) 37 (27.82) 70 (52.63) 7 (5.26) 133

*These two males were the parents in the two crosses (4 and 13) reported in Talavera et al. (1990).

(Talavera et al. 1990). All six males were mosaic for the pres- ence of E chromosomes in the testes, so that interfollicular variation was evident. However, all cells in a given follicle had the same number of E chromosomes. Table 1 shows the frequency of follicles with 0, 1, 2, or 3 E chromosomes in the six mosaic males. In all mosaic males, most follicles (about 85%) carried E chromosomes, and most of them (about 53%) possessed two E.

E chromosomes showed three important similarities with the X chromosome, namely, size (Fig. I), positive hetero- pycnosis in prophase, and autopairing (Fig. 2). Furthermore, E chromosomes associated frequently with X and M6 chro- mosomes (which are also heteropycnotic) during diplotene (Figs. 3 and 4), but these associations did not persist until metaphase I (Figs. 5 and 6). Table 2 shows the frequencies of association between these three heteropycnotic elements (X, E, and M6) during diplotene and metaphase I. Total frequency of diplotene X-E associations in follicles with one E (55.28%) was significantly higher than in follicles with two E (35.79%) (X2(l, = 7.42, P = 0.001-0.01). However, the frequency of diplotene X-M6 associations did not dif- fer significantly between one-E (30.89%) and two-E (27.37%) follicles (X2(l, = 0.17, P = 0.5-0.7). On the other hand, in two-E follicles the two E chromosomes were paired very frequently during prophase I (96.84%) and the association frequently persisted until metaphase I (48.44%) (Table 3). Two types of E-E associations were observed, namely, end to end and side by side, the former being more frequent and more persistent than the latter. Only X-E-E associations were observed; E-X-E ones, which could theoretically occur, were absent. Given the high frequency of E-E configurations, associations of a single E with the X were not observed either.

X-E segregation in the first meiotic division was studied by analyzing metaphase I1 cells. In follicles with one E, the E univalent had divided equationally in 6 cells and reduc- tionally in 65 cells: 27 with n = 8 + lE, 27 with n = 8 + X, 5 with n = 8, and 6 with n = 8 + X + 1E. If X and E segregated at random, 16.25 cells in each class would be expected. Chi-square tests demonstrated a signif- icant excess of cells derived from anaphase I cells in which X and E had segregated to opposite poles (xZo) = 28.48, P < 0.001). This indicates that X and E univalents did not segregate independently but moved to opposite poles in 76.06% of anaphase I cells. The X-E association of diplo- tene could predetermine that X and E will go to opposite poles in anaphase I. If this is so, this kind of segregation would be predicted in 34.15% + 21.14% + '/2 (44.71 %) = 77.64% (see Table 2). While 54 of 65 metaphase I1 cells analyzed were derived from anaphase I cells in which X and

E segregated to opposite poles, 1 1 were produced from X-E segregation to the same pole. The corresponding frequen- cies that would be expected according to the hypothesis that diplotene X-E associations predetermine their orientation towards opposite poles are 50.47 and 14.53, respectively. A x2 test did not rule out this hypothesis (X2(l,, = 1.10, P = 0.30-0.40).

In follicles with two E, only 6 of 150 metaphase I1 cells scored showed signs of equational division of one or both E chromosomes. Of 144 metaphase I1 cells derived from reductional division of E chromosomes, 17 contained n = 8 + X + 2 E , 2 6 n = 8 , 2 1 n = 8 + 2 E , 1 2 n = 8 + X, 32 n = 8 + X + lE, and 36 n = 8 + IE. The corresponding frequencies expected under random segrega- tion of X and E chromosomes would be 18, 18, 18, 18, 36, and 36, respectively. A X 2 test failed to demonstrate a sig- nificant difference between observed and expected numbers (x2(s, = 6.56, P = 0.2-0.3), which indicates random segregation of X and E chromosomes. Furthermore, the number of metaphase I1 cells observed with 0 (38), 1 (68), or 2 (38) E chromosomes did not differ significantly from those expected under independent segregation of the two E chromosomes (36, 72, and 36, respectively) (X2(2, = 0.44, P = 0.8-0.9).

Discussion The size and meiotic behavior of E chromosomes suggest

that they may have been derived from X chromosomes. E chromosomes are heteropycnotic and show autopairing during zygotene-pachytene, two general characteristics of X chromosomes in grasshoppers (John and Lewis 1965). Furthermore, X and E chromosomes associate during first prophase but do not do so at metaphase I, indicating that chiasmata are not formed. This seems to be a general feature of extra chromosomes (Hewitt and John 1968; Viseras and Camacho 1984), including B chromosomes (John and Hewitt 1965; Camacho et al. 1980) in grasshoppers.

The preferential segregation of X and E univalents to opposite poles during anaphase I fits well with the model for distributive pairing. X-E associations are not chiasmate since they do not persist until metaphase I, giving both chro- mosomes a second chance to segregate through distributive pairing. This is dependent on size, as Grell(1967, 1976) dem- onstrated in Drosophila that two nonhomologues of similar size segregate with the regularity of homologues, but as their sizes become more disparate the frequency of nondisjunc- tion increases significantly. X and E chromosomes of Ch. binotatus are very similar in size and do not form chiasmata, although they associate with each other during diplotene. Preferential segregation of X and E to opposite poles could

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LOPEZ-LEON ET AL.

FIGS. 1-6. Extra chromosomes (E) in the male germ line of Chorthippus binotatus. Fig. 1. Spermatogonial mitotic metaphase cell possessing two E chromosomes. Note that the size of an E chromosome is more similar to X than M4 chromosomes. Fig. 2. Early pachytene cell with one E chromosome autopaired and associated with the X. Figs. 3 and 4. Diplotene cells showing one E chromosome associated with the X. Figs. 5 and 6. Metaphase I cells with two E chromosomes. Note their association in Fig. 5 and their orientation to opposite poles in Fig. 6.

be predetermined by the X-E association during diplotene, ing, presumably late in prophase, at metaphase, or during and this hypothesis could not be ruled out by a X 2 test. anaphase of meiosis I. This is also true in Ch. binotatus, According to Grell (1964), meiotic chromosomes are in a since X and E chromosomes are well condensed from the well-condensed state when they undergo distributive pair- start of prophase I, but their association only persists until

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142 GENOME, VOL. 34, 1991

TABLE 2. X-E-M6 associations at diplotene and metaphase I

Follicle with Number (To) of cells showing different types of X-E-M6 associations extra

chromosomes Stage X-M6 X-E M6-E X-E-M6 No association Total

1E Diplotene 12(9.76) 42(34.15) l(0.81) 26(21.14) 42(34.15) 123 Metaphase I 0 0 0 0 161(100.00) 161

2E Diplotene 4(4.21) 12(12.63) 2(2.11) 22(23.16) 55(57.89) 95 Metaphase I 0 0 0 0 64( 100.00) 64

NOTE: The data for the different E-carrying males were pooled because o f the low numbers of cells available from each male separately.

TABLE 3. Associations between the two E chromosomes in follicles with 2E

Number (%) of cells showing E chromosomes

Stage End to end associated Side by side associated Nonassociated Total

Diplotene Metaphase I

NOTE: The data for the different E-carrying males were pooled because of the low numbers of cells available from each male separately.

diplotene. The way in which X-E association predetermines their regular segregation at anaphase I could be explained on the basis of the preorientation concept (Douglas 1968a, 1968b; Rickards 1983): centromere position established during prophase I predisposes the chromosomes to deter- mined orientations at prometaphase I. Thus if X and E are associated at diplotene and preorientation occurs, they could be predestined to segregate to opposite poles. Our scores of metaphase I1 cells were consistent with this hypothesis. Furthermore, the X chromosome in grasshoppers begins a back and forth movement from one to the other pole at pro- metaphase I, as has been demonstrated by in vivo studies of meiosis in Melanoplus differentialis (Nicklas 1961). If the X chromosome is associated to an E chromosome at the moment when this movement starts, and this latter is also capable of such movement, they could go to opposite poles in the first migration, and thus be predetermined to segregate. Another characteristic of distributive pairing is that when more than two elements are eligible, the pairing is competitive and preferential, depending on size (Grell 1964). When two E are present in Ch. binotatus, the X-E association at diplotene decreases by 20%, presumably due to the E-E pairing that occurs in almost all cells. Thus, the preference for the E-E pairing seems to produce a signifi- cant decrease in X-E associations. The segregation pattern observed in anaphase I is characterized by nondisjunction of both E chromosomes in 52.8% of cases, a fact that also agrees with the distributive pairing model: when nonhomol- ogous chromosomes pair distributively, abnormal segrega- tion patterns follow, which include nondisjunction of homologues and regular segregation of nonhomologues (Grell 1976). Although we are not sure whether X and E really share homology, there is no doubt that the two E chro- mosomes are homologues, so that their nondisjunction could be a reflection of distributive pairing.

Although the presumed distributive pairing of X and E chromosomes in Ch. binotatus may be based on their asso- ciation during prophase I, it is possible that it is a necessary but not a sufficient condition, in view of the contradictory results obtained in several species of grasshoppers where a nonrandom pattern of segregation between the X and B

chromosome has been detected. Thus, while in Tetrix ceperoi (Henderson 1961) and Phaulacridium vittaturn (Jackson and Cheung 1967) X and B moved preferentially to opposite poles (although in the latter John and Freeman (1974, 1975) and Rowe and Westerman (1974) found random segrega- tion after studying many populations), in Tettigidea lateralis (Fontana and Vickery 1973), Eyprepocnemis plorans (Camacho et al. 1980) and Dichroplus pratensis (Bidau 1986) X and B moved to the same pole (although in E. plorans this result was observed only in 1 population of the 5 analyzed). In all cases .the X-B associations were frequent at diplotene but rare at metaphase I. Thus, although this association could be presumed to be related to distributive pairing in Tetrix ceperoi and Ph. vittatum, given the pat- tern of regular segregation of nonhomologous chromosomes (similar to X-E chromosomes of Ch. binotatus), this is not the case in Tettigidea lateralis, E. plorans, and D. pratensis, where X-B pairing leads to increased nondisjunction of X and B chromosomes. Furthermore, the size requirement does not seem to be fulfilled, since the B chromosome of Tetrix ceperoi is much smaller than the X, while that of Ph. vittatum is larger than the X and the claim for preferential X-B segregation (Jackson and Cheung 1967) was not substantiated on reexamination (John and Freeman 1975). Thus other conditions besides prophase I association must be necessary for distributive pairing in grasshoppers. These conditions are fulfilled for E chromosomes in Ch. binotatus but not for B chromosomes in the above-mentioned species. Thus, distributive pairing seems to operate not only in Drosophila (Grell 1962) and yeast (Mann and Davis 1986; Dawson et al. 1986) but also in the grasshopper Ch. binotatus. Furthermore, distributive pairing could be responsible for the "pseudobivalent" formed between the X and Y chromatids at prophase I1 in the pentatomid plant bug, Nezara viridula, which ensures their disjunction to opposite poles (Camacho et al. 1985).

Acknowledgements This study was supported by the Spanish Direccion

General de Investigacion Cientifica y Tkcnica through Project PB87/0886, and the Plan Andaluz de Investigacion,

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LOPEZ-LEON ET AL. 143

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