Research Article |
Corresponding author: Bernard Dutrillaux ( bdutrill@mnhn.fr ) Academic editor: Robert Angus
© 2019 Anne-Marie Dutrillaux, Bernard Dutrillaux.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Dutrillaux A-M, Dutrillaux B (2019) Different behaviour of C-banded peri-centromeric heterochromatin between sex chromosomes and autosomes in Polyphagan beetles. Comparative Cytogenetics 13(2): 179-192. https://doi.org/10.3897/CompCytogen.v13i2.34746
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Heterochromatin variation was studied after C-banding of male karyotypes with a XY sex formula from 224 species belonging to most of the main families of Coleoptera. The karyotypes were classified in relation with the ratio heterochromatin/euchromatin total amounts and the amounts of heterochromatin on autosomes and gonosomes were compared. The C-banded karyotypes of 19 species, representing characteristic profiles are presented. This analysis shows that there is a strong tendency for the homogenization of the size of the peri-centromeric C-banded heterochromatin on autosomes. The amount of heterochromatin on the X roughly follows the variations of autosomes. At contrast, the C-banded heterochromatin of the Y, most frequently absent or very small and rarely amplified, looks quite independent from that of other chromosomes. We conclude that the Xs and autosomes, but not the Y, possibly share some, but not all mechanisms of heterochromatin amplification/reduction. The theoretical models of heterochromatin expansion are discussed in the light of these data.
Coleoptera, Polyphaga, karyotypes, heterochromatin, variation, sex chromosomes
There is a consensus to consider that the ancestral karyotype of Polyphagan beetles was composed of 20 chromosomes, a number observed in living specimens from most families (
We collected most of the specimens from the 344 studied species in France, Greece and West Indies. Some specimens were also obtained from amateur breedings, Besançon insectarium, or kindly provided by colleagues and friends. The species studied here were distributed into 21 families, but most belonged to Cerambycidae (67 species), Chrysomelidae (40 species), Curculionidae (18 species), Lucanidae (11 species), Scarabaeidae (136 species) and Tenebrionidae (28 species). We established the karyotype of the 344 species, among which we selected the19 following species, as examples of the various situations observed:
Adalia bipunctata Linneaeus, 1758 (Coccinelidae, Coccinelinae) (France);
Amphimallon solstitiale Linnaeus, 1758 (Scarabaeida, Melolonthinae) (France);
Asida jurinei Solier, 1836 (Tenebrionidae, Pimeliinae) (France);
Crioceris asparagi Linnaeus, 1758 (Chrysomelidae, Criocerinae) France;
Cyclocephala picipes Olivier, 1789 (Scarabaeidae, Dynastinae) (French Guyana);
Disonycha latifrons Schaeffer, 1919 (Chrysomelidae, Alticinae) (Canada, Quebec); Dorcadion (Cribridorcadion) etruscum Rossi, 1790 (Cerambycidae, Lamiinae) (Italy);
Lamprima adolphinae Gestro, 1875 (Lucanidae) (New Guinea);
Leucothyreus nolleti Paulian, 1947 (Scarabaeidae, Rutelinae) (Martinique);
Lilioceris lili Scopoli, 1763 (Chrysomelidae, Criocerinae) (France);
Lucanus cervus Linneaeus, 1753 (Lucanidae) (France);
Macraspis tristis Castelnau, 1840 (Scarabaeidae, Rutelinae) (Guadeloupe);
Melolontha melolontha Linnaeus, 1758 (Scarabaeidae, Melolonthinae) (France);
Melolontha hippocastani Fabricius, 1801 (Scarabaeidae, Melolonthinae) (France);
Morimus funereus Mulsant, 1862 (Cerambycidae; Lamiinae) (Greece);
Propomacrus davidi Deyrolle, 1874 (Scarabaeidae, Euchyrinae) (China);
Scarabaeus variolosus Fabricius, 1787 (Scarabaeidae, Scarabaeinae) (Greece);
Strategus syphax Fabricius, 1775 (Scarabaeidae, Dynastinae) (Guadeloupe);
Uloma retusa Fabricius, 1801 (Tenebrionidae, Tenebrioninae) (Guadeloupe).
After anaesthesia by ethyl acetate, testicular follicles were dropped into an aqueous solution of 0.88 M KCl where they remained for 15 min at room temperature. They were transferred into a micro-centrifuge tube (VWR International SAS, code 211-0033, Strasbourg, France) containing 0.5 ml of 0.55 M KCl (hypotonic) solution, where they were squashed and suspended using a piston pellet (VWR, code 045420) adjusted to the internal diameter of the tube. The volume of 0.55 M KCl was completed to 1.5 ml. After 10 min, they were centrifuged during 5 min at 800 g. The supernatant was replaced by Carnoy I fixative, in which the cells were suspended and left for at least 30 min. After one change of fixative, the cells were spread on wet and cold slides or conserved for a few days before use. Slides were stained by Giemsa, photographed and C-banded according to
Not all heterochromatin is stainable by C-banding, but for technical reasons, only C-band positive heterochromatin will be considered. The usual intra- and inter-specific variation of heterochromatin makes it somewhat arbitrary to decipher its amplification. At the level of the whole karyotype, we have visually considered that heterochromatin is not amplified (NAH) when its amount represents less than 10% of the total chromosome length (Fig.
For the above-mentioned species, this is the first report on C-banded karyotype, with the exception of L. cervus, L. adolphinae, M. tristis, M. hippocastani, M. melolontha and S. syphax (
Among the 344 male karyotypes studied, 25 (7.3%) without Y chromosome (X0 sex formula), 9 with a XYY formula (2.6%) and 35 (10.2%) with a gonosome-autosome translocation were excluded. Among the 275 remaining ones, the quality of the C-banding was considered to be sufficient for analysing both the size and the distribution of heterochromatin on chromosomes in 224 species. In this sample, a complete lack of C-banding on the Y chromosome was recorded in 134 instances (60%). At contrast, no C-banding was observed on the X chromosome in only 9 instances (4%). Among a large variety of profiles of heterochromatin distribution, some were particularly recurrent. They are listed below by order of decreasing occurrence.
a) Presence of clearly but not strongly amplified (NAH and MAH) C-banded heterochromatin on the centromere regions of all the chromosomes but the Y. It was observed in 86/224 instances (38.4%). Four examples are given in figure 1 in species from different families: A. bipunctata (Fig.
C-banded male karyotypes. A Adalia bipunctata B Asida jurinei C Morimus funereus D Cyclocephala picipes. The autosomes and the X chromosomes have similar amounts of C-banded heterochromatin, but the Y chromosomes remain unstained.
b) Presence of a clearly but not strongly amplified (NAH and MAH) C-banded heterochromatin on the centromere regions of all the chromosomes including the Y. It was observed in 60 instances (27%). Four examples are given in figure 2: A. solstitiale; D. etruscum; L. lili and S. syphax. Here again, there is some homogenization of the size of C-bands on both autosomes and X chromosome, but the size of the C-band on the Y is more independent: large in A. solstitiale (Fig.
C-banded male karyotypes. A Amphimallon solstitiale B Dorcadion etruscum C Lilioceris lili D Strategus syphax. In each karyotype, all centromere regions are similarly C-banded, but that of the Y is more variable.
c) Presence of large heterochromatic fragments (MAH and HAH) on both the autosomes and the X. It was observed in 28 instances (12.5%). In this condition, there is not a systematic homogenization of the heterochromatin size on the autosomes, as in U. retusa (Fig.
C-banded males karyotypes. A Uloma retusa B Lucanus cervus C Disonycha latifrons D Melolontha hippocastani. The level of heterochromatin amplification is often similar in the X and autosomes (A, B, D). The amplification may also be scattered, as in C, but it rarely involves the Y chromosome.
d) Presence of a large amplification of heterochromatin on the X chromosome but not on autosomes. It was observed in 25 species (11.2%). In these karyotypes, C-banded heterochromatin was either invisible on chromosome Y, as in L. nolleti and P. davidi (Fig.
e) Heterochromatin amplification on chromosome Y. It was noticed in 23 instances only (10.4%). Compared to both the X and autosomes, this amplification was almost always limited in size, some of the largest C-bands on the Y were observed in S. variolosus (Fig.
C-banded male karyotypes. A Leucothyreus nolleti B Propomacrus davidi C Lamprima adolphinae D Scarabaeus variolosus. Large heterochromatin amplification can involve the X alone (A, B, C) and more rarely the Y (D).
The analysis of most species was generally limited to a few specimens, but short series could be studied for some species. The high variability of both location and amount of heterochromatin is a common place, which was verified here. However, it appeared that variations of heterochromatin are more important on autosomes than on gonosomes. For example, amongst 18 males of M. melolontha, the X was always and the Y never C-banded. At contrast, the C-banding of several autosomes was highly polymorphic: it varied in size and could be either present or absent on a single or both homologs (Fig.
The possible heterogeneity of heterochromatin was investigated in the karyotype of C. asparagi, in which heterochromatin is strongly amplified on both the X and autosomes. As in most other species, its heterochromatin is homogenously stained after C-banding (Fig.
After BrdU incorporation during the late S-phase and acridine orange staining, heterochromatin homogenously fluoresces in orange, indicating its late replication, while early replicating euchromatin fluoresces in green (Fig.
Crioceris asparagi. A C-banded male karyotype displaying a large heterochromatin amplification in all chromosomes but the Y. B Incorporation of BrdU during late S-phase in a female cell: all heterochromatin is homogeneously late replicating (orange staining). The distal fragments of all chromosomes are early replicating (green), which indirectly indicates that there is no Lyonisation of one X. C C-banding of 3 spermatocytes (a, b, c) at pachynema : autosomal bivalents are at contact and form rosettes after heterochromatin fusion. The sex bivalent is always separated. D Q-banded male karyotype: heterochromatin displays at least 3 levels of fluorescence.
Structural chromosome rearrangements, such as reciprocal and Robertsonian translocations, fissions and intra-changes (inversions, translations, centromere shifts) recurrently occur and differentiate the karyotypes of related species. It seems that in beetles, in which most species possess 20 chromosomes, the karyotype diversification is principally the consequence of intra-changes, but this category of chromosome rearrangements remains difficult to detect, as long as chromosome banding is limited to heterochromatin (
The origin of heterochromatin and its highly repeated DNA content, as well as the factors modulating its quantitative and qualitative variations, remain largely unknown, but two main mechanisms have been envisaged.
1) The recombination process. As in other animals, peri-centromeric heterochromatin of beetles harbours sequences of repetitive (satellite) DNA (
2) The ocean ridges model. This model was proposed to explain the expansion of centromeric repeated DNA (
Most of the karyotypes of this report share the same tendency for heterochromatin homogenization. The more or less important heterochromatin or C-banding expansion is not totally independent from the systematic classification: for example, most Cerambycidae have small or inconsistent C-bands (Figs
In conclusion, there is a large variety of the heterochromatin patterns in the karyotypes of Polyphagan beetles. In spite of inter-individual variations, phylogenetically related taxa tend to share similar characteristics, but exceptions exist: huge amplifications of heterochromatin may affect only a single or all chromosomes of a karyotype and may characterize one or several species in a genus. Thus, heterochromatin constitutes a weak criterion for establishing phylogenetic relationships. A certain homogenisation of the heterochromatin amount and staining capacities exists between the autosomes of a same karyotype. The quantitative, but not qualitative, variations of the heterochromatin of the X grossly follow that of autosomes. At difference, the heterochromatin content of the Y is generally very limited and its variations look largely independent from those of other chromosomes. The concerted variations of autosomes, and the relative independence of the gonosomes, and the Y in particular, may be explained by the strong tendency for fusions of heterochromatin of autosomes, but not gonosomes, at male (and female?) meiotic prophase.