Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 72(2): 63-68, 2019 Firenze University Press www.fupress.com/caryologiaCaryologia International Journal of Cytology, Cytosystematics and Cytogenetics ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.13128/cayologia-194 Citation: A.-M. Dutrillaux, B. Dutrillaux (2019) Telomeric heterochromatin and meiotic recombination in three species of Coleoptera (Dorcadion olympicum Ganglebauer, Stephanorrhina prin- ceps Oberthür and Macraspis tristis Laporte). Caryologia 72(2): 63-68. doi: 10.13128/cayologia-194 Published: December 5, 2019 Copyright: © 2019 A.-M. Dutrillaux, B. Dutrillaux. This is an open access, peer-reviewed article published by Firenze University Press (http://www. fupress.com/caryologia) and distributed under the terms of the Creative Com- mons Attribution License, which per- mits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. Telomeric heterochromatin and meiotic recombination in three species of Coleoptera (Dorcadion olympicum Ganglebauer, Stephanorrhina princeps Oberthür and Macraspis tristis Laporte) Anne-Marie Dutrillaux, Bernard Dutrillaux* Systématique, Évolution, Biodiversité, ISYEB - UMR 7205 – CNRS MNHN UPMC EPHE, Muséum National d’Histoire Naturelle, Sorbonne Universités, 57 rue Cuvier CP50 F-75005, Paris, France *Corresponding author: bdutrill@mnhn.fr Abstract. Centromeres are generally embedded in heterochromatin, which is assumed to have a negative impact on meiotic recombination in adjacent regions, a condition required for the correct segregation of chromosomes at anaphase I. At difference, tel- omeric and interstitial regions rarely harbour large heterochromatic fragments. We observed the presence at the heterozygote status of heterochromatin in telomere region of some chromosomes in 3 species of Coleoptera: Dorcadion olympicum; Stephanor- rhina princeps and Macraspis tristis. This provided us with the opportunity to study the relationship between heterochromatin, chiasma location and meiotic recombination independently from the proximity of centromeres in this order of insects. In acrocen- tric chromosomes, the presence of heterochromatin in telomere region of the long arm displaces recombination near the centromere. In sub-metacentrics, recombination is almost always restricted to the other arm. This at distance effect of heterochromatin may deeply influence genetic drift. Keywords. C-banded, telomeric heterochromatin, meiosis, recombination, Coleoptera. INTRODUCTION In almost all living organisms, centromeres are surrounded by hetero- chromatin, which harbours repetitive DNA (Nakaseko et al. 1986), whose function is not yet completely understood. In cells in mitotic growth, het- erochromatin represses transcription and expression of genes located into it (Grewal and Jia 2007). During meiosis of most living organisms, recom- bination is necessary for the correct chromosome segregation at anaphase I, but it does not occur in heterochromatin. Consequently, recombination is repressed in centromeric regions, which harbour heterochromatin. In Schizo- saccharomyces pombe, it was found to be approximately 200 times less in het- 64 Anne-Marie Dutrillaux, Bernard Dutrillaux erochromatin than in the genome-wide average (Eller- meier et al. 2010). A current interpretation is that recom- bination, thus chiasma formation in centromeric region, would lead to abnormal chromosome segregation (Lynn et al. 2004). Thus, a function of centromeric heterochro- matin would be to displace recombination far from the centromeres and allow the correct chromosome segre- gation. Large and variable amounts of heterochromatin are present in the karyotype of many animals belonging to various taxonomic groups, but their position is not at random: frequently juxta-centromeric and rarely inter- stitial or terminal. In mammals, a wellknown example of terminal heterochromatin is that of the Hedgehog (Insectivora) in the karyotype of which 2 to 4 chromo- some pairs are involved. However, the heterochromatic blocks are not strictly terminal because NORs (Nucleo- lar Organizer Regions) are located at their extremity (Mandhal 1979). It was noticed that no meiotic recom- bination occurred in and at proximity of heterochroma- tin (Natarajan and Gropp 1971). The same particularities were observed in the Primate Cebus capucinus (Dutril- laux 1979) but such examples remain rare. In insects, terminal heterochromatin was observed in acrocentric chromosomes of several species of grasshoppers (John and King 1982, 1985, Torre et al. 1985). These authors attributed the displacement of chiasmata to proximal position to the presence of terminal heterochromatin. In Coleoptera, large heterochromatic fragments are com- monly seen in most families (Juan and Petitpierre, 1989, Correa et al., 2008, Dutrillaux and Dutrillaux, 2016), but almost always in the centromere region, as in other taxonomic groups. This preferential location may result from the amplification of DNA repeated sequence sur- rounding centromeres, as shown for α satellite sequences (Rudd et al. 2006, Shepelev et al. 2009). As it will be dis- cussed, the correct segregation of chromosomes at meio- sis may also depend on the embedding of centromeres in heterochromatin. Displacement of heterochromatin from centromere regions to intercalary or terminal regions would necessitate secondary events, but terminal het- erochromatin may have other origins. It will be also dis- cussed that the presence of heterochromatin in telomeric position may not confer a selective advantage, by impos- ing meiotic constraints. Among hundreds of species of Coleoptera we studied, karyotypes with large amounts of heterochromatin in terminal position were rarely observed. We confirm that heterochromatin terminally located on the long arm of acrocentrics may not decrease recombination, but simply displaces it near to the cen- tromere, usually considered as a cold region (Mahtani and Willard 1998). In addition, we show that in non- acrocentric chromosomes, heterochromatin terminally located on one arm displaces recombination to the other arm. Thus, heterochromatin can suppress recombination on a whole arm and influence meiotic recombination at much larger distance than it was generally thought. MATERIAL AND METHODS Three examples belonging to three different families or sub-families were found among about 400 species of Polyphagan beetles: Dorcadion olympicum Ganglebauer 1882 (Ceram- bycidae: Lamiinae: Dorcadionini). Two specimens were captured in May 2014 in Eastern Greece, near Alexan- droupolis (40° 50’57”N and 25°52’46”E). Stephanorrhina princeps Oberthür 1880 (Scarabaei- dae: Cetoninae: Goliathini). Two specimens of African origin (Malawi) were obtained in September2007 from a private breeding. Macraspis tristis Laporte 1840 (Scarabaeidae: Ruteli- nae: Rutelini). Eight adult specimenswere obtained in March 2008 from grubs captured in Guadeloupe (Basse- Terre, near Deshayes 16°18’00”N and 61°47’00”W) in December 2006. Chromosome preparations of cells at various stages of meiosis were obtained as described (Dutrillaux and Dutrillaux 2009, Dutrillaux et al. 2010). Proliferating cells obtained from either eggs, testes or mid gut were processed as described. Chromosomes were Giemsa stained and further silver stained for localization of the Nucleolus Organizer Region (NOR) and/or C-banded for localization of heterochromatin. Image capture and karyotyping were performed using IKAROS software (Metasystems, Germany). Chromosome nomenclature: to avoid ambiguous interpretations, we will call acro- centric all chromosomes with a single euchromatic arm, whatever the size of the heterochromatin (generally C-banded) forming the other arm. Chromosomes with euchromatin (not C-banded) on both arms are either metacentric or sub-metacentric. We will focus on chro- mosomes with large heterochromatic blocks distally attached to euchromatic arms. RESULTS Dorcadion olympicum The mitotic male karyotype is composed of 24 chro- mosomes. Pairs 3 and 5 are sub-metacentric and all other autosomes are acrocentric. The X chromosome is sub-metacentric and the Y is punctiform (24,XY). In one of the two specimens studied, the length of one chromo- 65Telomeric heterochromatin and meiotic recombination in three species of Coleoptera some 11 is enlarged by the addition of a large amount of C-banded heterochomatin at the telomeric region of the long arm. (Fig. 1a). Following a simple Giemsa staining, the chromatids of this fragment look hyper-cohesive, thin and pale. Centromeric regions are faintly C-band- ed, as it frequently occurs in the genus Dorcadion (per- sonal data). At diakinesis/metaphase I of meiosis, the sex bivalent has the parachute configuration, usually found in Polyphagan Coleoptera. The compaction of the addi- tional heterochromatin of bivalent 11 is much variable: quite elongated at early diakinesis, it becomes highly compacted at late metaphase I. Bivalent 11 remains eas- ily identified by C-banding (Fig. 1b). The euchromatic component of bivalent 11 has the same aspect in the 131 analysed metaphases I: a cross with very uneven branch- es. The block of heterochromatin is always located at the tip of the longest branch. This indicates that chiasmata are systematically located near the centromere. In 90/95 spermatocytes II at metaphases, the heterochromatic block is located on a single chromatid of chromosome 11, demonstrating that one crossing-over had occurred in its long arm (Fig. 1c). In 5 instances only, the hetero- chromatin is present on both chromatids, which may be interpreted as either a lack of recombination in the long arm or the result of a double recombination between the centromere and the heterochromatin. Interestingly, in these chromosomes, the heterochromatin blocks remain cohesive, while euchromatic arms are well separated, as usual at this stage. This gives it a ring appearance (Fig.I d). Finally, in 4 additional pairs of sister spermatocytes II (or diploid ones), the heterochromatin carrier chro- mosomes remain close to each other suggesting hetero- chromatin remained associated at anaphase, inducing chromosome lagging. Stephanorrhina princeps The mitotic karyotype is composed of 18 meta- or sub-metacentric autosomes, one large X, and one punc- tiform Y (20, XY). Large and variable fragments of het- erochromatin are C-banded on one chromosome of pairs N°5, 6, 8 and on the X. In pair N°5, the hetero- chromatin is distally located on the short arm (Fig. 2a). We will focus on the meiotic behaviour of the heterozy- gote chromosomes 5. As in the previous species, the chromatids are hyper-cohesive in their heterochromatic portion. At diakinesis/metaphase I, the heterochroma- tin is fuzzy and hardly detectable without C-banding. In 37/40 instances, the heterochromatin is located at one extremity of bivalent 5, which looks asymmetrical after C-banding (Fig. 2b). In 3 instances, heterochromatin is in the centre of the bivalent, which has a symmetri- cal appearance. Amongst 23 spermatocytes II at meta- phase, chromosomes 5 have a C-band on either both chromatids (12 times), or a single chromatid (twice, fig. Fig. 1. Chromosomes of Dorcadion olympicum : a) Giemsa-stained karyotype and C-banded chromosomes 11 exhibiting additional heterochromatin in one homologue. b) Giemsa stained (left) and C-banded (right) spermatocyte I at diakinesis/metaphase with biva- lent 11 carrying heterochromatin at one side (arrow). X and Y form a parachute bivalent (Xyp). Barr= 10 mm, as in other figures. c) Group of 3 spermatocytes II at metaphase, sequentially Giemsa stained (G) and C-banded (c). As in most other metaphases II, centromeric het- erochromatin is poorly C-banded and chromosome 11 is asymmetri- cal, with compacted heterochromatin at the tip of a single chromatid (arrows). d) 12,Y spermatocyte II in which the 2 heterochromatin carrier chromatids of chromosome 11 remain cohesive, whereas all other chromosomes have clearly non-cohesive chromatids. Fig. 2. Sequentially Giemsa stained (G) and C-banded (C) chromo- somes of Stephanorrhina princeps a) Giemsa stained (center) and C-banded karyotype of a spermatogonium exhibiting additional heterochromatin on one chromosome of pairs 5, 6, 8 and on the X. Only heterochromatin of chromosome 5 is clearly separated from the centromere region. b) Diakinesis/metaphase I: heterochromatin (arrows) remains opposite to chiasmata. c) Exceptionally, in each of these 2 brother spermatocytes II, chromosome 5 is asymmetrically carrier of heterochromatin. d) Spermatocyte I at pachynema: synap- sis defect (SD) of the proximal (euchromatic) part of the short arm of bivalent 5. 66 Anne-Marie Dutrillaux, Bernard Dutrillaux 2C), or had no C-band (9 times). Thus, recombination rarely occurred in the heterochromatin carrier arm. At pachynema, the bivalents with enlarged heterochromatin tend to remain close to each other, in spite of the drastic hypotonic shock to which they had been submitted. A synapsis defect of the euchromatic fragment comprised between the telomeric and centromeric heterochromatin was recurrently observed (Fig. 2d). Macraspis tristis The karyotype, composed of 18 chromosomes, is characterized by the frequent presence of large and vari- able heterochromatic fragments at telomeric regions of one arm of 3 pairs of sub-metacentrics (N°6, 7 and 8) and on the X (Fig. 3a). In all diakineses/metaphases I examined from all the specimens, the heterochromatin carrier bivalents have the same configuration: chiasma or terminal association in the euchromatic arms and opposite position of the heterochromatin (Fig.3b). This suggests that no recombination occurred in the hetero- chromatin carrier arms. In the specimen considered here, pairs N°7 and 8 were heterozygote for the pres- ence or absence of a large heterochromatin fragment. Among 11/12 spermatocytes II, all carrier chromosomes had similarly heterochromatin in both chromatids (Fig. 3c). In a single one, one chromosome was asymmetri- cal, with heterochromatin on a single arm, indicating that recombination took place between the centromere and the heterochromatin (Fig. 3d). In a second speci- men, only pair N° 8 was heterozygote for the presence of heterochromatin. No asymmetry was observed on chro- mosomes from 28 spermatocytes II. Finally, in a third specimen, both pairs N° 7 and 8 were heterozygote, and no asymmetry was detected among 8 spermatocytes II. Thus, in 65 analysed sub-metacentrics, recombination was almost always suppressed between the distally locat- ed heterochromatin and the centromere, and occurred in the other arm (64/65 times). As in S. princeps, the bivalents with enlarged heterochromatin tended to asso- ciate at pachynema. DISCUSSION Recombination and heterochromatin Only few data exist on meiotic recombination and chromosome segregation in beetles, and most of them were obtain before the heterochromatin detection was possible (Smith and Virkki 1978). In both literature and our own data, a general observation is that most biva- lents exhibit a single chiasma in a fairly distal, if not terminal, position at diakinesis/metaphase I. This is in agreement with the findings, in other organisms, that repression of recombination occurs not only in juxta-cen- tromeric heterochromatin, but also in adjacent regions. Thus, recombination hot spots are rarely located near to the centromeres, but most frequently in intercalary and near telomeric regions (Lichten and Goldman, 1995). The presence of large heterochromatin fragments in chromo- somes is not exceptional. They generally occur in centro- meric regions, where recombination rarely occurs (Eller- meier et al., 2010). Their more exceptional occurrence at telomeric regions offers the possibility to look for the possible influence of heterochromatin on meiotic recom- bination and chromosome segregation, independently from the proximity of the centromere and associated repetitive DNA. Such analysis was already performed in grasshoppers, in which some species have terminal het- erochromatin in acrocentric chromosomes. It showed the displacement of chiasmata to a proximal position in het- erochromatin carrier, compared to other chromosomes (John and King, 1982, 1985, de la Torre et al., 1986). Our observations in D. olympicum confirm these findings: at metaphase I, the heterochromatic block of chromosome 11 is almost always at distance from the chiasma. In addition, we could quantify recombination through the analysis of 95 spermatocytes at metaphase II: 90/95 chromosomes 11 carry heterochromatin on a sin- gle chromatid, which formally demonstrates an almost systematic occurrence of crossing over between the cen- tromere and the heterochromatic block. Thus, there was Fig. 3. Giemsa (G) stained and C-banded (C) chromosomes of Macraspis tristis. a) C-banded karyotype of a spermatogonium exhibiting heterozygosity for heterochromatin of pairs 7 and 8 and homozygosity for pairs 5 and 6. b) Sequentially Giemsa stained and C-banded spermatocyte I at metaphase with asymmetric bivalents 7 and 8. Heterochromatin of bivalents 5, 6, 7 and 8 is always external, opposite to chiasmata or terminal association. c) Spermatocyte II: all heterochromatin fragments (arrows)are symmetrically distribut- ed on both chromatids. d) Unique spermatocyte II with asymmetri- cal chromosome 7. 67Telomeric heterochromatin and meiotic recombination in three species of Coleoptera no crossing-over suppression but a displacement towards proximal regions. The 5 metaphases II with symmetrical chromosomes 11 cannot be interpreted univocally: either 2 or no crossing-over occurred or crossing-over occurred in the short (heterochromatic) arm. As discussed below, these few cells provide us with interesting information about chromosome cohesion and segregation. In M. tristis and S. princeps, all autosomes are sub- metacentric, and the analysis of spermatocytes I at metaphase indicates that most bivalents form a single chiasma. For chromosomes with one heterochromatin carrier arm, chiasmata are almost systematically located on the other arm. This absence of recombination in the heterochromatin carrier arm is confirmed by the analy- sis of spermatocytes II in metaphase: at difference with the acrocentric of Dorcadion, heterochromatin is almost always either present or absent on both arms. This sug- gests a “choice” between the two arms, by suppression of recombination in the whole heterochromatin carrier arm. As observed in a proportion of pachytene cells of S. princeps, there is an asynapsis of the whole euchromatic fragment located between centromeric and telomeric heterochromatin, which may be related to the lack of recombination. These 3 examples show that large telom- eric heterochromatin fragments can drastically influence meiotic recombination in euchromatin, with an effect at a long distance. It may create a hot spot of recombina- tion fairly close to the centromere, which is quite unusu- al, as in D. olympicum. It may also generate a cold and a hot arm, as in the two other species. This effect is prob- ably not due to the heterozygote status, because when both arms are carrier, they are generally not involved in chiasmata at diakinesis. By altering meiotic recombina- tion, these occasional heterochromatic fragments alter the gene linkage between all the genes of the chromo- some and influence genetic drift. Cohesion, compaction and heterochromatin At metaphase I, sister chromatids are maintained together by cohesins, a ring-shaped protein complex formed by 4 sub-units: Scc1 (Rec8), Scc3, Smc1 and Smc3. At anaphase I, cohesins are cleaved by separase all along chromatids, except in centromere regions where the sub-unit Rec8 is protected from cleavage by the protein complex Shugoshin/protein phosphatase PP2A, which counteracts its phosphorylation (Riedel et al. 2006). This permits the resolution of chiasmata, which occur in euchromatic fragments. At the following meta- phase II, cohesins are no more efficient. Chromatids are well separated and chromosome cohesion is maintained at the centromere regions only. Finally, after inactivation of PP2A, centromere cleavage occurs at anaphase II, ena- bling the segregation of monochromatidic chromosomes. Why and how Shugoshin/PP2A complex is located in centromere regions remains unknown. Our observation may provide some information. In spermatocytes II, all chromosomes have well separated chromatid, with the exception of chromosomes 11 of D. olympicum, which are ring shaped when they are symmetrically carrier of terminal heterochomatin. It means that cohesin was not cleaved both at centromere and telomere, the two het- erochromatic regions. Thus, Rec8 seems to be protected from cleavage by heterochromatin. This protection, nec- essary for the correct chromosome segregation at ana- phase I, may be one of the reasons why centromeres are systematically embedded in heterochromatin. It is commonly observed that in metaphases of mitotic cells also, heterochromatic fragments are more cohesive than euchromatin. At the molecular level, the search of a particular relationship between the protein complexes involved in cohesion and heterochromatin components could be of interest. The DNA methylation status may play a role in this context. In human cells, although juxta-centromeric heterochromatin of chromo- somes 1, 9 and 16 is not G-C rich, it is strongly labelled by antibodies to 5-MdC (5-methyldeocycytidine), indi- cating its strong methylation status (Miller et al., 1974; Montpellier et al., 1994). In mouse germ cells, large vari- ations of DNA methylation were reported during the progression of gametogenesis (Coffigny et al. 1999; Ber- nardino-Sgherri et al. 2002; Marchal et al. 2004). Drastic changes from hypo- to hyper-methylation occur in het- erochromatin and euchromatin in an opposite way. For example, in early spermatogonia, hypo-methylated and elongated centromeric heterochromatin displays prema- ture cleavage, while chromosomes remain cohesive at hyper-methylated chromatids. On the opposite, euchro- matic chromatids are cleaved in spermatocytes II, when they are poorly methylated while chromosomes remain cohesive at their methylated and compacted centromere regions. Unfortunately, we could not study the methyla- tion status of beetle chromosomes during gametogenesis, but the high similitude of variations of chromosome compaction and cohesion suggests they might correlate with DNA methylation changes. In conclusion, the presence of large fragments of heterochromatin at telomere regions deeply alters mei- otic recombination in beetles, as in other animals. 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