Heredity 79 (1997) 135—143 Received 26 June 1996

Kinetic organization of metaphase I bivalents in spermatogenesis of and Trichoptera species with small chromosome numbers

KLAUS WERNER WOLF-I-, KAREL NOVAK & FRANTIEK MAREC* tlnstitut für Anatomie der Medizinischen Universität, Ratzeburger A/lee 160, D-23538 Lübeck, Germany and /nstitute of Entomology, Czech Academy of Sciences, Braniovská 31, CZ-37005 Ceské Budejovice, Czech Republic

Thefine structure of bivalents in metaphase I spermatocytes of two Lepidoptera species, thyellina Butler (n =11)and 0. antiqua (L.) (n =14)(Lymantriidae), and a Trichoptera species, Limnephilus decipiens (Kolenat) (n =10)(Limnephilidae) were studied using a series of ultrathin sections and transmission electron microscopy. The bulk of species in both orders possess a haploici chromosome number of about 30. Thus, the experimental specieshave relatively small chromosome numbers. This study showed that metaphase I bivalents in both Lepidoptera species are polykinetic; attachment of kinetochore microtubules is found scattered throughout the entire poleward chromosomal surface. The microtubules were inserted in material of medium electron density. A pair of distinct kinetochore plates, consisting of material of about the same electron density as the chromatin, was detected at each poleward chromosomal surface in metaphase I bivalents of the caddis-fly, L. decipiens. The observations suggest that DNA elements responsible for the organization of the kinetochores are dispersed throughout the chromosomes in the two Lepidoptera species, whereas they are narrowly clustered in the Trichoptera. Thus, karyotype evolution in the closely related Lepidoptera and the Trichoptera involved widely differing mechanisms.

Keywords:karyotypeevolution, kinetochores, meiosis, microtubules.

ochores were found at each poleward surface of the Introduction chromosomes. The kinetochores consisted of plates Theinsect orders Lepidoptera and Trichoptera are of medium electron density that were in contact with considered sister groups on the basis of about 20 microtubules (MTs). Ephestia kuehniella (Pyralidae) synapomorphies, common characteristics derived and B. mon (Bombycidae) have a haploid chromo- from a common ancestor (Kobayashi & Ando, 1988; some number of 30 and 28, respectively (Traut & Kristensen,1991; Neboiss, 1991; Nielsen & Mosbacher, 1968; Traut, 1976; Holm & Rasmussen, Common, 1991). Several synapomorphies also apply 1980). The caddis-fly, A. furcata (Limnephilidae), to properties of the karyotype. In both orders, has not been precisely karyotyped, but published females are the heterogametic sex and female micrographs of a chromosome spread suggest that meiosis is of the achiasmatic type (Suomalainen, the haploid chromosome number is around 30 (fig. 5 1966; Traut & Mosbacher, 1968). Fine structure in Wolf et al., 1992). Similarity between representa- studies involving two Lepidoptera species, the Medi- tives of both groups with about 30 chromo- terranean flour Ephestia kuehniella (Wolf & somes is not restricted to chromosome structure in Traut, 1991) and the silkworm Bombyx mon (Holm meiosis. In metaphase spermatogonia, kinetochore & Rasmussen, 1980), and a Trichoptera species, plates cover around 40 per cent of the chromosomal Anabolia furcata (Wolf et a!., 1992) revealed that the length (Gassner & Klemetson, 1974; Wolf et al., architecture of bivalents in metaphase I spermato- 1992). cytes is very similar. In all cases, two distinct kinet- Information on the kinetic organization of chromosomes in Lepidoptera and Trichoptera Correspondence. E-mail: [email protected] species with exceptionally small chromosome

1997 The Genetical Society of Great Britain. 135 136 K.WWOLFETAL. numbers is scarce. In any case, electron microscopy atmosphere for dissection to Lübeck. In this species, is required because Lepidoptera chromosomes are which undergoes imaginal diapause in native condi- unusually small (Goodpasture, 1975). In a previous tions (Novak & Sehnal, 1963), the larvae approach- fine structure study, the presence of polykinetic ing pupation appeared to be optimal for preparation metaphase I bivalents has been mentioned in Orgyia of meiotic chromosomes in metaphase I (cf. Le thyellina Butler (Lymantriidae), a moth with a Lannic, 1975). haploid set of 11 chromosomes (Wolf et al., 1987). In other words, MT contacts were found scattered throughout the entire poleward surface of the Preparationfor e/ectron microscopy chromosomes. The emphasis of the study was, Thepreparation procedure has been described however, on the comparison of eupyrene and previously (Wolf, 1994). In brief, the dissected apyrene meiosis and chromosome structure was not gonads were transferred to Ringer solution (Glaser, documented in detail. In brief, Lepidoptera show 1917: cited by Lockwood, 1961) containing 2.5 per double spermatogenesis. Eupyrene meiosis gives rise cent glutaraldehyde. The composition of the Ringer to spermatids which transform into fertilizing sperm, solution was: 9 g NaC1, 0.42 g KC1, 0.25 g CaCI2 and whereas apyrene meiosis leads to a non-fertilizing 0.2 g NaHCO3 per litre doubly distilled water. After sperm variety (Meves, 1903; Wolf, 1994). In the 5 mm, three volumes of 8 per cent tannic acid present article, which is only concerned with eupy- (Merck) in phosphate buffer (0.067 M, pH 6.8) were rene meiosis, we dwell on the kinetic organization of added. The gonads were postfixed in phosphate- metaphase bivalents in primary spermatocytes of 0. buffered 0s04 (1 per cent), dehydrated in ethanol thyellina. In addition, we present information about and embedded in Epon 812. Ultrathin sections, bivalent structure in eupyrene spermatogenesis of about 70 nm thick, were mounted on Pioloform- another species of the same genus, 0. antiqua (L.), coated grids, doubly stained with uranyl acetate and which possesses a haploid Set of 14 chromosomes lead citrate and analysed with a Philips EM 400 (Cretschmar, 1928). Also in this species, spindle fine transmission electron microscope operated at 80 kV. structure has been previously described (Wolf, In 0. thyellina, one longitudinally sectioned and 1990), but information on the kinetic organization of one cross-sectioned metaphase I plate have been the bivalents is missing. Because only eupyrene sper- recorded using series of consecutive sections. In 0. matocytes are involved, we dispense with this attri- antiqua and L. decipiens, it has been one longitudi- bute in the text when Lepidoptera are addressed. nally sectioned metaphase I spindle in each. In all The findings in the Lepidoptera species are cases, primary spermatocytes were studied, and the compared with those in a Trichoptera species, findings were confirmed by inspection of about six Limnephilus decipiens (Kolenat) (Limnephilidae), neighbouring cells of the same developmental stage with a haploid set of 10 chromosomes (Klingstedt, and taken from the same cyst. 1931; for a compilation of chromosome numbers in the Trichoptera, see Lankhorst, 1970). Results Materials and methods Orgy/a thye//ina An/ma/s The fine structure of metaphase I spermatocytes of 0. thyellina has been described previously. In brief, Wild-type last instar larvae of 0. thyellina and 0. the spindle area was surrounded by a multilayered antiqua were kindly provided by Sir Cyril A. Clarke sheath of membranes structurally reminiscent of (Liverpool, UK) (cf. Wolf et a!., 1987; Wolf, 1990). agranular (smooth) endoplasmic reticulum (ER). Prior to preparation, the were maintained Membranous elements were also scattered through- for several days in the laboratory and fed with out the spindle area. The spindle poles were marked hawthorn (Crataegus oxyacantha). by pairs of basal bodies continuous with flagella Second to third instar larvae of L. decipiens were (Wolf et a!., 1987). In the present study, the kinetic collected in 'Domin' pond near Ceské Budéjovice in organization of bivalents was analysed in a serially May 1993, maintained in an aquarium and fed with cross-sectioned metaphase I spindle. A total of 11 reed leaves (Phragmites communis), lettuce leaves chromosomes could be identified as individuals and (Lactuca sativa), and dead cricket larvae (Giyllus they were very similar. Serial cross-sections through domesticus). At the beginning of June, fully grown the poleward surface of one bivalent, situated in the larvae were placed in the refrigerator to prevent spindle periphery, are depicted (Fig. la—i). At least pupation, and then transferred in a highly humid five major kinetochore MT bundles were in contact

The Genetical Society of Great Britain, Heredity, 79, 135—143. KINETOCHORES IN LEPIDOPTERA AND TRICHOPTERA 137

Fig.1 (a—i) Transmission electron micrographs of nine consecutive serial sections through the poleward chromosomal surface of a metaphase bivalent of Orgyia thyellina. The section plane is at right angles to the spindle axis. The proximity of the spindle envelope (SE), which consists of membrane stacks, signals that the bivalent lies in the spindle periphery. Several major bundles of microtubules (arrowheads) penetrate into the chromatin. Note cross-sectioned spindle micro- tubules and intraspindle membranes lateral to the bivalent. (Bar represents 0.5 zm.)

The Genetical Society of Great Britain, Heredity, 79, 135—143. 138 K. W. WOLF ETAL. with the poleward chromosomal surface. The MTs ochore plates laid in shallow recesses of the chroma- penetrated up to seven sections, about 500 nm, into tin. Although they showed about the same electron the chromatin. Material of lesser electron density density as the chromatin, the kinetochore plates was found at the MT ends. The MT ends were scat- could be readily distinguished, because they were tered throughout the chromosomal surface. Thus, separated by a transparent gap from the underlying the previous assessment that bivalents in male chromatin. The kinetochore plates were disc-shaped meiosis of 0. thyellina are polykinetic (Wolf et al., with a diameter of 200—250 nm. An inner kineto- 1987) has been confirmed. chore layer, as we know it from mammalian kinet- ochores (cf. Rieder, 1982), was, however, not observed in bivalents of the caddis-fly. The kinet- Orgy/aant/qua ochore plates were in contact with MT bundles. The Theoverall architecture of metaphase I spermato- chromosomal surfaces, laterally located with respect cytes of 0. antiqua and, in particular, the structure to the kinetochore plates, were largely devoid of MT of the spindles were very similar to that in 0. thyel- contacts. lina (cf. Wolf, 1990). There was a prominent layer of perispindle membranes and alsointraspindle membranes were abundant. Longitudinal serial Discussion sections through metaphase I spindles were inspec- Bivalentstructure in male meiosis of Lepidoptera ted in order to determine the kinetic organization of and Trichoptera species with chromosome numbers the bivalents. These never showed lateral contacts around 30 is very similar. Each poleward chromo- with one another. Independent of the size, the somal surface possesses two individual kinetochore chromosomes were very similar. MT bundles were plates and MTs are connected with the material. attached to the poleward chromosomal surfaces. A The kinetochore plates are, however, slightly less preferential location of the MT ends was not detect- distinct in the Lepidoptera (Holm & Rasmussen, able. The MT ends were scattered throughout the 1980; Wolf & Traut, 1991; Wolf et al., 1992). Lepi- entire surface. The MT ends were embedded in doptera and Trichoptera are considered monophy- material of lesser electron density. At several sites, letic sister groups (Kobayashi & Ando, 1988). It is threads of electron-dense chromatin extended for therefore a strong possibility that the common 400—600 nm from the chromosomal surfaces ancestors of the Lepidoptera and Trichoptera shared (Fig. 2a—l). As in 0. thyellina, this indicates that the the bivalent architecture described above. A simi- MTs penetrate into the chromatin. larity of the commonest haploid chromosome numbers, n =31in the Lepidoptera and n =30in Limnephilus the Trichoptera, was interpreted as another synapo- dec/p/ens morphic trait of the sister groups (Suomalainen, Theanalysis of serial longitudinal sections through 1966, 1969). Indeed, chromosomal variance in the metaphase I spindles in testes of the caddis-fly Lepidoptera shows a marked mode at 28—3 1 revealed a conventional bipolar spindle apparatus. throughout most families (Suomalainen, 1969; Spindle-associated membranes were scarce. Only Robinson, 1971; White, 1973). Representatives of few membranous sheets reminiscent of the smooth the most primitive lepidopteran families do not ER were found. In contrast with the Lepidoptera differ from the others in this respect (n 31—32; species described above, the bivalents showed a few Suomalainen, 1969). Identification of the type lateral contacts. Nevertheless, most chromosomes chromosome number in the Trichoptera appears, could be identified as individuals. Independent of however, incorrect. Kiauta (1971) argued that the the sizes of the bivalents the kinetic organization number of n =30cannot be regarded as the true was very similar. As a rule, the chromosome surfaces type number of Trichoptera, although it was most were relatively smooth. The individual bivalents frequently reported. In fact, the n =30complement possessed two distinct kinetochore plates at each is typical of the most advanced family Limnephilidae poleward surface, representing the kinetic apparatus but has not been found in any other family exam- of two sister chromatids. Thus, each bivalent showed ined so far (see Lankhorst, 1970). On the other a total of four kinetochore plates. In small bivalents, hand, relatively high chromosome numbers, ranging the centre-to-centre distance between the individual from n =22to n =30,predominate in most studied kinetochore plates of the sister chromatids was families of the suborder Integripalpia, although low about 600 nm (Fig. 3a—d). In large bivalents, this numbers also occur. The other suborder, Annuli- distance was up to 1.3 tm (Fig. 4a—d). The kinet- palpia, with only three species karyotyped shows low

The Genetical Society of Great Britain, Heredity, 79, 135—143. KINETOCHORES IN LEPIDOPTERA AND TRICHOPTERA 139

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4* L IT1t of Orgyia Fig.2 (a—I) Transmission electron micrographs of 12 consecutive serial sections through a metaphase bivalent antiqua. The section plane is in parallel to the spindle axis and the bivalentis situated in the spindle periphery as indicated by the spindle envelope (SE). Material of lesser electron density than the chromatinis found at various sites on both chromosomal surfaces (arrowheads). Microtubules are in contact with the material. (Bar represents 0.5 tim.) chromosome numbers, n =13and n =15(see the in the Trichoptera, could have repeatedly evolved as Ross' phylogenetic tree of Trichoptera in Kiauta, a derived character in different families. 1971). Taken together, it is likely that the common The divergence of the two orders involved a series ancestor of the Trichoptera and Lepidoptera had a of changes. Higher Lepidoptera, for example, form high haploid chromosome number close to 30. two morphologically and functionally different Hence, low numbers of chromosomes, in particular sperm types (Meves, 1903; Wolf, 1994), whereas the

The Genetical Society of Great Britain, Heredity, 79, 135—143. 140 K. W. WOLF ETAL.

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Fig.3 (a—d) Transmission electron micrographs of four consecutive serial sections through a small metaphase I bivalent of Limnephilus decipiens. The section plane is in parallel to the spindle axis. A pair of kinetochore plates (arrowheads) is attached to each poleward chromosomal surface. (Bar represents 0.5 tim.) most primitive Lepidoptera and all Trichoptera well as their irregular surface in longitudinal produce only one sperm type (Friedländer, 1983; sections. We suggest that the polykinetic organiza- Sonnenschein & Häuser, 1990; Hamon & Chauvin, tion in the two Orgyia species examined could evolve 1992). At the cellular level, meiotic spindles in male from originally localized kinetochores of their Lepidoptera show an abundant set of spindle-assoc- ancestor with the haploid chromosome number iated membranes, whereas the Trichoptera spindles around 30; such numbers were reported for three have a relatively poor membrane inventory. The other species of the same genus, 0. ericae and 0. abundance of intraspindle membranes, which is recens (n =30)and 0. leucostigma (n =28)(see typical of male meiosis in the Lepidoptera (Wolf, Robinson, 1971). Chromosome fusions most prob- 1995), may be responsible for the strict separation of ably took place in the evolution of the small the individual bivalents in metaphase I spindles of chromosome numbers of 0. antiqua and 0. thyellina 0. thyellina and 0. antiqua. The poor intraspindle (cf. Suomalainen, 1969). Then, polykinetic organiza- membrane system of L. decipiens renders it possible tion of the neochromosomes could be established that chromosomes laterally associate with one provided that the chromosome fusions were not another. followed either by further rearrangements leading to The present study showed another diverging cellu- fusions of kinetochore plates or inactivation of addi- lar trait: the kinetic organization of metaphase I tional kinetochores. In contrast to the two Lepidop- bivalents in male meiosis of representatives with tera, metaphase I bivalents of the Trichoptera small chromosome numbers. The bivalents of two species, L. decipiens, with a haploid set of 10 Lepidoptera species, 0. thyellina and 0. antiqua with chromosomes showed two distinct kinetochores n =11and 14, respectively, were polykinetic. MTs located in shallow recesses at each poleward were found connected with the entire poleward chromosomal surface. The two sites represent the chromosomal surface. Attachment sites of MTs kinetochores of two sister chromatids (for a scheme, consisted of material of lesser electron density than see Fig. Sb). A very similar kinetic organization has the chromatin, and were situated in relatively deep been described in another limnephilid, A. furcata, recesses of the chromosomal surface. Distinct kinet- which has, however, n30 (Wolf et al., 1992). The ochore plates as shown in E. kuehniella (Gassner & high number of chromosomes is characteristic for Klemetson, 1974; Wolf & Traut, 1991) were, Limnephilidae, found in five of the 10 examined however, not detected. Our interpretation of the species of the genus Limnephilus; whereas it varies polykinetic organization is illustrated in Fig. 5a. from 6 to 16 in the other five species (Lankhorst, Each chromatid carries several attachment sites 1970; Kiauta, 1971). One can, therefore, expect that which are distributed throughout their surface. In the low chromosome number in L. decipiens repre- the metaphase I bivalent, the chromatids are prob- sents, similar to 0. antiqua and 0. thyellina, a ably wound or somehow folded to expose the attach- derived character. In this case, however, genome ment sites to MTs. This arrangement could explain rearrangements leading to the reduction of chromo- the roughly spherical appearance of the bivalents as some number did not result in a polykinetic organi-

The Genetical Society of Great Britain, Heredity, 79, 135—143. KINETOCHORES IN LEPIDOPTERA AND TRICHOPTERA 141 tt

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Fig.4(a—d)Transmission electron micrographs of four consecutive serial sections through a large metaphase I bivalent of Limnephilus decipiens. The section plane is in parallel to the spindle axis. A pair of kinetochore plates (arrowheads)is attached to each poleward chromosomal surface. (Bar represents 0.5 tm.) zation of the chromosomes. Thus, in karyotype detected in this group, is increasing (for a review, evolution of the Trichoptera the putative ancestral see Wolf, 1996). As a rule, the centromere-assoc- type of kinetic organization (see above) was iated DNA is repetitive. It is plausible to assume conserved, while the Lepidoptera developed a novel that species-specific repetitive DNA is also involved in the formation of kinetochores in the Lepidoptera type. In mammalian cells, kinetochores are organized and Trichoptera. The highly diverging appearance of by specific DNA elements (Choo et a!., 1991) and the kinetochores in the species examined in the this appears to be the case also in . The present study suggests far-reaching differences in the number of centromere-specific DNA sequences, distribution of the underlying DNA along the

The Genetical Society of Great Britain, Heredity, 79, 135—143. 142 K.W.WOLFETAL.

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ChJ' MTs , BBH/H: liii! liii i'i f Fig.5 Schematic drawings of two metaphase I bivalents with different kinetic organization. (a) A proposed structure of a bivalent with polykinetic organization, the situation found in two Lepidoptera species, Orgyia thyellina and 0. antiqua. (b) Stnicture of a bivalent with the putative ancestral type of kinetic organization (see the text for details), showing localized, relatively large kinetochores. A and A', B and B', the poleward surface of two sister chromatids of two homologous chromosomes; AS, attachment site of microtubules; Ch, chromatin; K, kinetochore plate; MTs, microtubules of the spindle. chromosomes. It is clustered in the Trichoptera and vation of centromeres, which has been observed in highly dispersed in the Lepidoptera. Thus, karyotype dicentric chromosomes of mammals (Earnshaw & evolution in the Lepidoptera and Trichoptera Migeon, 1985), can be ruled out in the present case, differed widely. This is also apparent in the varia- because MTs were associated with both kineto- bility of the chromosome numbers. Whereas chores. Trichoptera show moderate variation —thesmallest and the highest chromosome numbers are 6 and 30, Acknowledgements respectively, (Kiauta, 1971) —theLepidoptera have a larger range in chromosome number than any Theauthors are grateful Mrs S. Glatzel and Mrs F. Niedereichholz for excellent technical assistance. other group (from n =7to n =c.220) (Robinson, 1971; White, 1973; cf. also Wolf, 1994). The work was supported by the 'Deutsche Thus, dispersion of centromere-specific DNA is Forschungsgemeinschaft' (Wo 394/6—1). FM was likely to render chromosomes more susceptible to or supported by the grant no. A607105 and its renewal tolerant of karyotype rearrangements. no. A6007609 of the Czech Academy of Sciences. The appearance of the large metaphase I bivalents in L. decipiens is informative, because the individual References kinetochore plates at the poleward chromosomal AULT,J. G. AND LYTTLE, T. w. 1988. A transmissible dicen- surface were up to 1.3 m apart from one another. tric chromosome in Drosophila melanogaster Chromo- Principally the chromosomes are dicentric. Although soma, 97, 71—79. transmissible dicentric chromosomes have been C1-100, K. H., vissEL, 8., NAGY, A., EARLE, E. AND KALITSIS, described (Ault & Lyttle, 1988; Wandall, 1994), this P. 1991. A survey of the genomic distribution of alpha condition is usually correlated with a high rate of satellite DNA on all the human chromosomes, and chromosome breakage and segregation. The reason derivation of a new consensus sequence. Nuci. Acids for this behaviour lies in the risk that the two kinet- Res., 19, 1179—1182. ochores of one poleward surface become oriented CRETSCHMAR, M. 1928. Das Verhalten der Chromosome bei der Spermatogenese von Orgyia thyellina Btl. und towards opposite spindle poles in metaphase. antiqua L., sowie eines ihrer Bastarde. Z. Zellforsch. Spindle forces consequently tear the chromosome Mikrosk. Anat., 7, 291—399. apart. In the case of the Trichoptera chromosomes, EARNsHAw, w. C, AND MIGEON,B. R. 1985.Three related however, either the distance between the individual centromere proteins are absent from the inactive kinetochores is too small and/or the chromosomes centromere of a stable isodicentric chromosome. Chro- are too stiff to allow this kind of orientation. macti- mosoma, 92, 290—296.

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