Heredity7O (1993) 135—141

Genetical Society of Great BritainReceived 13Apr11 1992

The relationship between synapsis andchiasma distribution in grasshopper bivalents

heterozygous for supernumerary segmentsJ. L. SANTOS, A. DEL CERRO, A. FERNÁNDEZ & M. D1EZ

Departamento Genética, Facultad de Biologla, Liniversidad Complutense, 28040 Madrid, Spain

The relationship between chiasma distributions and pairing behaviour of grasshopper bivalentheterozygous for supernumerary segments has been analysed. The extra segments were located atthe telomeric end of the long arm in chromosome S8 of Chorthippus jacobsi and at interstitialposition in chromosomes S8 and M7 of Euchorthippus pulvinatus. In all cases these heteromorphicbivalents exhibited fuiiy paired synaptonemal complexes with equalized axial lengths either at latezygotene or early pachytene, that is, heterosynapsis in regions near the extra segments is achieved atthese meiotic stages. It is proposed that the absence of chiasmata in those regions in which non-homologous pairing took place, would lead to chiasma redistributions observed in these bivalentswhen compared with those observed in bivalents in which extra segments are lacking.

Keywords: chiasma distribution, meiosis, supernumerary segments, surface spreading, synapsis.

Introduction

Populations of grasshoppers frequently show differen-tial polymorphism with respect to the presence andfrequency of supernumerary segments on one or moremembers of the standard complement. This extramaterial is in most cases of heterochromatic nature andis located on small chromosomes in proximal, inter-stitial or distal regions.

The main effect of these segments is at the endo-phenotypic level, mainly on the recombination process.In fact, they can increase the mean cell chiasma fre-quency of the male carriers when compared to non-carriers (see for instance Hewitt & John, 1968; Riva etal, 1984) and produce changes in chiasma distributionof the bivalents bearing them in such a way that regionsfurthest from the segment are preferred for chiasmaformation (Miklos & Nankivell, 1976; John & King,1985; Navas-Castillo eta!., 1985).

It has recently been proposed that the redistributionof chiasmata in bivalents with polymorphic ends couldbe produced by the misalignment of homologouschromosomes where they are attached to the nuclearenvelope (Moens eta!., 1989). In this paper we provideexperimental data on chiasma distributions and pairingbehaviour of bivalents heterozygous for super-numerary segments and bivalents without such seg-ments in two species of grasshopper, Chorthippusjacobsi and Euchorthippus pulvinatus.

135

Materials and methods

Testes of three males heterozygous for a super-numerary segment in the S8 chromosome (BS) and offive males without the segment (basic homozygotes,BB) of Chorthippus jacobsi and testes of one maleheterozygous for a segment in the M7 chromosome(BS), of two males heterozygous for a segment in the S8chromosome and of three basic homozygotes ofEuchorthippus pulvinatus were used in this study. Theywere selected from last instar nymphs collected at theCampus of Complutense University (1990) andValdemoro (Madrid, 1990) respectively.

Testes were divided into two parts, one fixed inethanol—glacial acetic acid (3:1) and stored at 4°C.Preparations were made in 45 per cent acetic acid and,after removing the coverslips, slides were air dried andstained by the C-banding procedure described bySantos & Giráldez (1978). The other part was sub-jected to a surface spreading procedure according tothe method of Croft & Jones (1986), with some modifi-cations. Briefly: testes, without the fat body, werehomogenized in Ham's F12 medium. Two drops ofdetergent solution (0.03 per cent Joy in distilled water,pH = 7.5) were added to a drop of homogenized testissuspension on a cavity slide, left for 5 mm and washedonto plastic-coated slides with seven drops of para-formaldehyde fixative (pH =8.9). The preparationswere then dried overnight on a warm plate at 30—35°C,

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136 J. L. SANTOS ETAL.

rinsed and air dried. For silver impregnation, a fewdrops of 50 per cent AgNO3 solution were placed onthe slide and covered with a patch of nylon cloth.Nuclei were examined using a Zeiss EM 902 andphotographed on Kodak 4489 film.

The position of the single chiasma in M7 and S8bivalents was classified by eye as proximal, interstitialor distal with respect to the centromere. Data fromdifferent individuals with the same C-banding patternfor the chromosome analysed (M7 or S8) have beenpooled because they showed similar patterns ofchiasma distribution.

Results

Chorthippus jacobsi

As in other gomphocerine grasshoppers, this specieshas 17 chromosomes (16 + X) in the male and 18(16 + XX) in the female. Three pairs of autosomes arelong and submetacentric and the remaining five pairsof autosomes and the X-chromosome are of medium tosmall size and acrocentric. Giemsa C-banding revealedcentromeric heterochromatin in all chromosomes andtelomeric heterochromatin in M5, M6, M7 and S8chromosomes. Quantitative heterochromatin variationwas observed in the M7 chromosome. In M5, M6 andS8 chromosomes telomeric heterochromatin was alsovariable in size and could even be absent. Figure 1shows a metaphase I cell in which a S8 heteromorphicbivalent is observed.

One chiasma is invariably formed in S8 bivalentsindependently of their heterochromatin constitution.The chiasma distributions in two classes of S8 biva-tents, homozygotes without the segment (BB) andheterozygotes (BS) are shown in Fig. 2. In S8 BB biva-lents, chiasmata were preferentially located at inter-stitial and distal positions whereas in BS bivalents an

increase in proximal and interstitial chiasmata and aparallel decrease in distal ones was observed.

Analysis of zygotene surface spread nuclei in thisspecies, as in other grasshoppers, is complicated due todifficulties in tracing axial and lateral elements becausethey are very thin and in many cases they can bestretched, overlapped or interlocked. Shorter bivalents(M6, M7 and S8) complete pairing quite early andshow full pairing in zygotene cells where about 30 percent of the complement is asynapsed. When paired,these shorter bivalents display similar synaptonemalcomplex (SC) lengths (J. L. Santos et al., unpublishedobservations). For this reason S8 BB and S8 BSindividuals, chosen for the analysis of pairing pro-gression, were homozygotes, with or without C-hetero-chromatin, for M6 and M7 chromosomes.

To estimate the position of unpaired regionsthroughout M6-S8 bivalents in S8 BB and S8 BSindividuals, paired and unpaired regions were

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Fig. 2 Chiasma distributions in the S8 bivalent ofChorthippusjacobsi. (a) basic homozygotes (b) hetero-zygotes.

GRASSHOPPER BIVALENTS 137

measured in zygotene nuclei, expressed as percentagesof bivalent lengths and plotted on the standard karyo-type derived from pachytene SC measurements (J. L.Santos ci a!., unpublished observations). The resultsobtained are shown in Fig. 3. In S8 BB males, the endsof the short arms of M6-S8 bivalents were usuallyasynapsed and in some nuclei the centromeres too,indicating that pairing starts at or near the telomericregion of the long arms. However, two pairing initiationpoints, located at both ends of long arms, have alsobeen observed. In S8 BS individuals one bivalentdisplaying unequal axis lengths was observed whichrepresents the S8 bivalent heterozygous for the super-numerary segment. In this bivalent the pattern of pair-ing initiation changes completely because it starts at ornear the short arm and proceeds towards the telomericend of the long arm (Fig. 4 a—c). In two nuclei thelonger axial element showed a region of fold-back pair-ing (Fig. 4b). When pairing progresses the differencesin length between axial elements decrease (r =—0.87,d.f. =6) in such a way that at late zygotene (70—80 percent pairing) M6-S8 bivalents exhibit fully pairedsynaptonemal complexes with equal axis lengths (Fig.4d). This implies that in these bivalents a variableproportion of non-homologous pairing is achievedbefore pachytene where crossings-over occur.

Euchorthippus pu/vinatus

The karyotype of this species is similar to that reportedfor Ch. jacobsi in terms of chromosome number andmorphology but it shows a different C-banding pattern.

Fig. 3 The relative lengths and positions of paired regions(thin lines) in M6-S8 bivalents of Cli.jacobsi from mid-latezygotene nuclei plotted on the pachytene SC karyotype. (a)Basic homozygotes for S8 chromosome (BB). Data takenfrom J, L. Santos et al. (unpublished results). (b) Segmentheterozygotes for S8 chromosome (BS).

Thus, the M7 chromosome can have a thick C-bandnear the telomere in the Valdemoro population (Santos& Giráldez, 1982) and the S8 chromosome can have athin interstitial C-band near the centromere.

M7 and S8 bivalents are invariably monochiasmate.The chiasma distributions in two classes of M7bivalents (BB and BS) are shown in Fig. 5. In M7 BSbivalents chiasmata tend to be located close to thecentromeric regions (Fig. 6a), although a relatively highfrequency of distal associations, which are of chiasmatenature (Santos & Esteban, 1990), have also beenobserved (Fig. 6b). In contrast, in M7 BB bivalentschiasmata are located all along the chromosome.

Analysis of silver-stained SCs in M7 RB bivalentsindicated that synapsis at zygotene is either unidirec-tional from the telomeres of the long arms towards thecentromeric regions and the short arms or bidirectionalfrom initiation at or near both telomeres. Among theshorter bivalents, M7 can be identified because thereare considerable differences in length with respect toM6. On the other hand, the S8 bivalent carries anucleolar organizer region.

Only seven nuclei of the M7 BS male could beanalysed: three were at late zygotene with 79, 86 and90 pairing percentages respectively and the other fourwere at pachytene with haploid SC lengths of 972, 957,902 and 885 m. In two of the zygotene nuclei (79 and90 per cent paired) and in one of pachytene (957 tm),both ends of M7 bivalent were synapsed resulting in abuckle comprising axes of unequal length. The size ofthe buckle was 14 and 11 per cent of the longer axislength in the zygotene nuclei (Fig. 7a—b) and 4 per centin the pachytene nucleus (Fig. 7c—d). Then, it isapparent that the size of the loop decreases from zygo-tene to pachytene. Its distance to the nearest telomereis 23, 18 and 4 per cent of the longer axis lengthrespectively. In the remaining nuclei straight pairingcan be observed (Fig. 7e). We conclude that, as in Cli.jacobsi, certain chromosome regions of this bivalentshow non-homologous pairing before or at the pre-sumed time of chiasma formation.

No surface spread zygotene nuclei from the twoindividuals heterozygous for the S8 chromosome wereobtained. All pachytene nuclei analysed (10) revealedstraight-paired synaptonemal complexes with equallengths. Therefore, heterosynapsis for the interstitialC-band is achieved at or before this stage.

Discussion

The electron microscopic analysis of SCs revealed thatin the grasshopper species, analysed here, the hetero-zygosity for supernumerary segments changes thenormal pattern of synapsis within the bivalents carrying

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Fig. 4 Electron micrographs of silver stained SC preparations of Ch. jacobsi showing patterns of synapsis in the heteromorphicS8 bivalent. (a) Zygotene SC exhibiting unequal axis lengths; the paired region (between arrows) corresponds to that near thecentromere. (b) Zygotene SC with fold-back pairing in the longer axis. Regions showing homologous and non-homologoussynapsis are arrowed. (c) Zygotene SC with asynapsed portions of the pairing axes of unequal length (arrowed). (d) Pachytene SCshowing full pairing and equalized axes. C =centromere, T= telomere. Bars represent 2.5 1um.

them. In S8 BB bivalents of Chorthippusjacobsi males,synapsis usually starts at or near the distal ends of longarms; however, in S8 BS bivalents synapsis is initiatedat or near the distal ends of short arms and proceeds tothe ends of the long arms in an unidirectional fashionfollowed by heterosynapsis involving the additionalC-heterochromatic segment (Fig. 3). It is feasible that,as has been observed in Chloealtis conspersa (Moens et

at., 1989), misalignment of heteromorphic cores andsubsequent synaptic failure as a consequence of theirattachment to the nuclear envelope can be involved inthis process.

At late zygotene S8 BS bivalents show equalizedaxes. Therefore, a certain degree of non-homologouspairing in the proximity of the extra segment takesplace before pachytene. Cross-over formation between

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GRASSHOPPER BIVALENTS 139

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Fig. 6 C-banded diplotenes from a E. pulvinatus male heterozygous for a supernumerary segment in M7bivalent. (a) M7bivalent showing a proximal chiasma (arrowed). (b) M7 bivalent showing a distal chiasma (arrowed).

strictly homologous regions, mostly proximal andinterstitial ones, would lead to the chiasma distributiondisplayed by these bivalents (Fig. 2).

Comparable situations involving an accommodationmechanism, which functions to equalized the lengths ofthe two axes of the heterochromatic synaptonemalcomplexes, have been reported in deer mice hetero-zygous for the presence of heterochromatic short arms(Hale & Greenbaum, 198 8a,b). In contrast, no evi-dence for an adjustment effect reducing the telomerelength differences throughout pachytene—diplotenewas found in rye bivalents heterozygous for telomericC-bands. Furthermore, heterozygosity for the 1RLterminaiC-band did not modify the chiasma distribu-

tion pattern of this arm (Gihies & Lukaszewski, 1989).The large number of pairing initiation sites in plantscompared to that displayed by the short chromosomesof rodents and grasshoppers may provide an explana-tion for these contradictory results.

Light microscope observations in plants of Scillasiberica, heterozygous for interstitial C-bands, indi-cated that heterochromatin interferes with euchromaticpairing at later stages of pachytene leading to the for-mation of a loop produced by the non-pairing adjacenteuchromatic segment. Berger & Greilhuber (1991)concluded that this segment would be excluded fromrecombination. On the other hand, SC observations inplants of rye heterozygous for an interstitial C-band on

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Fig. 7 Electron micrographs of silver stained SC preparations of E. pulvinatus showing patterns of synapsis in the hetero-morphic M7 bivalent. (a) Detail of the buckle in (b). (b) Zygotene bivalent showing a buckle configuration (arrowed), both endsare synapsed. (c) Detail of the buckle in (d). (d) Pachytene bivalent showing a partially adjusted buckle (arrowed). (e) Pachytenebivalent showing axes with equal length and absence of buckle. Bars represents 2.5 m.

chromosome 6R indicated the existence of hetero-synapsis following alignment of the C-band region andthese heteromorphic bivalents showed a chiasmaredistribution when compared with non C-bandcarriers (L. E. Benavente, unpublished results).

Similar results have been reported in animals, thusheterosynapsis at an interstitial C-band heterozygositytook place in the red kangaroo Macropus rufus (Sharp,1986), in the rodent Peromyscus beatae (Sudman et al.,

1989) and in the grasshopper Euchorthippuspulvinatus (this work). Whereas the dynamics of theprocess is unknown in the S8 bivalent of the latterspecies, it is clear that in the M7 bivalent synapsis isbidirectional from both telomeres leading to a buckleformation. If we consider the size and the position ofthe buckle (Fig. 7b and d) it appears that it can beformed in different locations in the bivalent. This con-clusion is based on the observation that the maximum

GRASSHOPPER B!VALENTS 141

distance observed from the buckle to the nearest telo-mere is higher than the maximum size loop. On thecontrary, if the buckle was always formed at the sameposition and subsequently readjusted, an increase inthe distance from the buckle to the telomere would beexpected during this process but this has not beenobserved. Therefore, a certain extent of pairing aroundthe buckle must be non-homologous. This hypothesisis reinforced when the euchromatin—heterochromatinratio in M7 mitotic chromosomes is considered (about1:1), although heterochromatin may be underrepre-sented in the SC (Stack, 1984). Nevertheless, by pachy-tene and in some nuclei by late zygotene the lateralelements of the heteromorphic bivalent display thesame length. As chiasmata are formed between homo-logous chromosome regions and a variable proportionof non-homologous pairing involves the C-band loca-tion and surroundings, one would expect a high fre-quency of proximal and distal chiasmata, as observed(Fig. 5).

In conclusion, the heterozygosity for interstitial andtelomeric supernumerary segments produce differentvariations in the synaptic patterns of the carrier biva-lents when compared with non-carriers. However, atlate zygotene or early pachytene heterosynapsis isachieved in both cases which implies a certain degreeof non-homologous pairing. The absence of chiasmatain regions near the segments, those in which non-homologous pairing took place, would lead to thechiasma distributions observed in these heteromorphicbivalents. In this context, the dependence of the inten-sity of chiasma redistribution on the size of the extrasegment found by Navas-Castillo et al. (1987), couldbe explained on the basis that small size segments pro-duce less non-homologous pairing in bivalents carryingthem than large ones.

AcknowledgementsThis paper was supported by a grant PB 88-0121awarded by Comision Interministerial de Ciencia yTecnologia (Spain). Dr Nieves Cuñado, José Barrios,Agustin Fernández and the staff of the ComplutenseUniversity Electron Microscope Unit, especially DrCarlos Barba, provided valuable assistance.

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