A South American origin of the genus Dynastes (Coleoptera: Scarabaeidae: Dynastinae) demonstrated by chromosomal analyses.

The karyotypes of 5 species of Dynastes, D. hercules, D. tityus, D. granti, D. satanas, and D. neptunus, and 2 subspecies of D. hercules are compared with those of 6 other selected Dynastinae species. In the 3 former species, there are 18 chromosomes, including neo-sex chromosomes formed by the fusion of an acrocentric autosome with the X and Y chromosomes. In all other species, including D. neptunus and D. satanas, free X and Y chromosomes are observed in 20,Xyp karyotypes. The acrocentric autosome presumably involved in the fusion is present in D. neptunus and D. satanas (pair No. 8). It replaces a submetacentric observed in the other Dynastinae species. Thus, the karyotypes of D. neptunus and D. satanas derive from that of ancestral Dynastinae by an inversion in chromosome 8, and those of D. hercules, D. granti and D. tityus derive from that of D. neptunus by the translocation of this inverted chromosome to sex chromosomes. Because the Dynastes species with the most derived karyotype occur in North and Central America and Lesser Antilles, while D. neptunus and D. satanas are limited to the northern part of the South American Andes, we suggest a South American origin of the genus Dynastes.

Low, complex and probably reticulated chromosome evolution of Sciuromorpha (Rodentia) and Lagomorpha.

Lagomorpha (rabbits and pikas) and Sciuromorpha (squirrels) are grouped in the Glires superorder. Their chromosome diversification, since their separation from the eutherian mammalian common ancestor, was characterized by a low rate of chromosome rearrangements. Consequently, the structure of some chromosomes was either conserved or only slightly modified, making their comparison easy at the genus, family and even order level. Interspecific in situ hybridization (Zoo-FISH) largely corroborates classical cytogenetic data but provides much more reliability in comparisons, especially for distant species. We reconstructed common ancestral karyotypes for Glires, Lagomorpha, Sciuromorpha, and Sciuridae species, and then, determined the chromosome changes separating these ancestors from their common eutherian ancestor. We propose that reticulated evolution occurred during the diversification of Glires, which implies that several pericentric inversions and Robertsonian translocations were conserved in the heterozygous status for an extensive period. Finally, among Lagomorpha and Sciuromorpha, we focused on Leporidae and Sciuridae chromosome evolution. In the various attempts to establish dichotomic evolutionary schemes, it was necessary to admit that multiple homoplasies (convergent and reverse rearrangements) occurred in Sciuridae and in a lesser degree, in Leporidae. In Leporidae, additional rearrangements were sufficient to propose a resolved phylogeny. However, a resolved phylogeny was not possible for Sciuridae because most of the rearrangements occurred in terminal branches. We conclude that a reticulated evolution took place early during the evolution of both families and lasted longer in Sciuridae than in Leporidae. In Sciuridae, most chromosome rearrangements were pericentric inversions involving short fragments. Such rearrangements have only mild meiotic consequences, which may explain the long persistence of the heterozygous status characterizing reticulated evolution.

Chromosome analysis of 82 species of Scarabaeoidea (Coleoptera), with special focus on NOR localization.

The aim of this study was the identification of the ancestral location of the nucleolus organizer region (NOR) in the Scarabaeoidea superfamily, and its evolutive trends in the karyotypes. For this purpose, the mitotic and meiotic chromosomes at pachynema of 82 species belonging to 4 families and 8 subfamilies, including 49 species without any published data, were examined after Giemsa staining, C-banding and NOR staining. It could be perceived that most karyotypes are composed of 18 nonacrocentric autosomes, an acrocentric X and a punctiform Y. NORs are frequently located on the X independent of its morphology. In contrast, autosomal NORs are frequently on the rare acrocentric short arms. Thus, it could be shown that the ancestral karyotype was very probably composed of 18 metacentric/submetacentric autosomes, an NOR carrier acrocentric X and a punctiform Y. The NOR translocation on autosomes parallels the passage to their acrocentric morphology. It is proposed that the frequent location of the NOR on the X of beetles, and possibly other insects, is made possible by their mode of dosage compensation of the X chromosome, consisting in the overexpression of the unique X of the males.

Cyclocephala (Coleoptera: Scarabaeidae: Dynastinae) evolution in Lesser West Indies indicates a Northward colonization by C. tridentata.

A dual cytogenetic and molecular analysis was performed in four species of Cyclocepala (Coleoptera: Scarabaeidae: Dynastinae) from Lesser Antilles (Martinique, Dominica and Guadeloupe). Two species/sub-species, C. mafaffa grandis and C. insulicola, are endemic to Guadeloupe. They have their own non-polymorphic karyotype and a fairly homogeneous haplotype of the COI gene. C. melanocephala rubiginosa has a distinct karyotype. Its COI haplotype is homogeneous in Guadeloupe and heterogeneous in Martinique. Finally, C. tridentata has highly different karyotypes and haplotypes in the three islands. In Martinique, its karyotype, composed of metacentrics, is monomorphic while its haplotype is fairly heterogeneous. Both are close to those of other Cyclocephala and Dynastinae species, thus fairly ancestral. In Guadeloupe, its karyotype is highly polymorphic, with many acrocentrics, and its haplotype fairly homogeneous. Both are highly derived. In Dominica, both the karyotype and the haplotype represent intermediate stages between those of Martinique and Guadeloupe. We conclude that several independent colonization episodes have occurred, which excludes that C. insulicola is a vicariant form of C. tridentata in Guadeloupe. Both chromosome and COI gene polymorphisms clearly indicate a recent colonization with a northward direction for C. tridentata.

Conserved although very different karyotypes in Gliridae and Sciuridae and their contribution to chromosomal signatures in Glires.

Rodents represent the largest order of living mammals. It comprises 5 sub-orders, among which Sciuromorpha (Sciuridae, Gliridae and Aplodontiidae) are assumed to occupy a basal position in rodent evolution. Banded karyotypes of some representatives of the Sciuridae family have been compared to each other, and comparisons with man were performed using chromosome paintings. Sciuridae karyotypes have conserved several eutherian ancestral syntenies. Like Sciuridae, Gliridae possess some chromosomes easily comparable with those of Primates. Comparisons of Gliridae and Sciuridae chromosomes with those of the presumed eutherian ancestor provide information about their chromosomal evolution and their position among Rodentia. Although both Sciuridae and Gliridae karyotypes are relatively conserved, they display many differences, indicating their early divergence. The reconstruction of their chromosomal evolution allowed us to propose the composition of their presumed ancestral karyotypes, with 2n = 48 and 2n = 38 for Gliridae and Sciuridae, respectively. Since rodent emergence, a single rearrangement is common to these 2 families. It formed a chromosome with fragments homologous to human chromosomes 4-8p-4-12-22, not detected in other rodents, and thus characteristic for the Sciuromorpha. This allowed us to reassess the chromosomal signatures of Rodentia. Finally, we show that the speed of chromosomal evolution in Gliridae is intermediate between that of Sciuridae (low) and Muridae (high).

Evolution of European cockchafers (Melolonthinae: Scarabaeidae: Coleoptera): a morphological, molecular and chromosomal study of intra- and inter-specific variations.

In cockchafers of the genus Melolontha, there is a marked intraspecific polymorphism for morphological characters, making some specimens of one species resemble another. A cytogenetic and molecular (mitochondrial COI gene sequence) study of typical and atypical forms of M. melolontha and M. hippocastani, captured at the same period and area, was performed. Karyotypes and haplotypes clearly characterize each taxon, placing atypical specimens in one or the other species unambiguously. This formally discards the role of hybridization in phenotypic resemblance, as usually proposed. Karyotypes and haplotypes were compared to those of M. pectoralis and Phyllophaga pleei, a more distantly related Melolonthinae, and some Dynastinae species, to reconstruct their ancestral karyotype. The karyotype of M. melolontha is the most derivative and that of P. pleei the most conserved among the Melolonthinae studied, which fits with the phylogeny established by COI gene analysis. Both karyotypes and COI haplotypes demonstrate the proximity of M. pectoralis and M. melolontha. The karyotype of M. melolontha is polymorphic, without relationship with morphological variations. Finally, the existence of similar morphological variations in different Melolontha species and chromosomal polymorphism in M. melolontha is discussed in relation with a network (reticulated) mode of speciation.

Y-chromosome disomy and trisomy in scarabaeid and cerambycid beetles.

In a series of about 500 specimens, including 420 males, of karyotyped Polyphaga beetles, 5 males with chromosome Y aneuploidy were detected. One male of each Dicronorrhina derbyana oberthuri (Scarabaeidae), Agapanthia violacea and Morimus funereus (Cerambycidae) were XYY, and 2 probably related and sterile males of Marmylida marginella (Scarabaeidae) were XYYY. These and literature data suggest that Y chromosome aneuploidies are much more frequent in polyphagan beetles than any other group of animals with an XY/XX sex determinism. The origin of this particularity probably lies in the unique mode of sex chromosome association at meiosis I: it is not synaptic but realized through nucleolar proteins forming the well-known parachute-like structure (Xy(p)). This has 2 possible consequences. The first one is the regular association of several sex chromosomes at metaphase I and segregation at anaphase I. It allows, for instance, XYY (Xyy(p)) males to procreate XYY sons. The second consequence is the occasional remain of nucleolar proteins embedding sex chromosomes in spermatocytes II. We propose that it could impede the correct segregation of Y chromatids after centromere split at anaphase II, and contribute to form YY gametes by XY males and YYY gametes by XYY males. The tendency for increasing the number of Ys would not be strongly limited at the XY level, but only at the XYY level by male infertility at higher Y ploidies.

Chromosome polymorphism and Fanconi-like instability in the scarabaeid beetle Macraspis tristis from Guadeloupe.

The karyotype of Macraspis tristis Laporte is described. It is composed of 18 chromosomes. C-band positive heterochromatin is very abundant and is located at centromeric regions and, for some chromosomes, at telomeric regions. There is a high inter-individual chromosomal polymorphism for the presence and extension of telomeric heterochromatin. In one of the 8 specimens studied, 2 populations were observed in testicular cells. Besides groups of normal metaphases, other groups displayed multiple chromatid and chromosome alterations such as breaks, exchanges (radials), deletions and translocations, resembling those described in Fanconi anemia. The insect had a normal phenotype, but its gametogenesis did not reach the spermatocyte II stage, and was quite poor in spermatocytes I. The clonal character of the chromosome instability was obvious owing to the incomplete cytodiaeresis of germ cells which remain associated by cytoplasmic bridges. This may be the first example of chromosome instability observed in animals from nature.

Sex chromosome rearrangements in Polyphaga beetles.

The presence of a parachute sex chromosome bivalent (Xyp) at metaphase I of male meiosis is a well-known characteristic of Coleoptera, present in almost all families of this order and assumed to represent their ancestral sex chromosome formula. Sex chromosomes appear to be manifold more frequently involved in inter-chromosomal rearrangements than the average of the nine autosomal pairs usually forming their karyotype. This leads to various formulae such as neo-sex, multiple sex and perhaps unique sex chromosomes. These rearrangements alter the intimate association between sex chromosomes and nucleolar proteins, which are usual components of the Xyp. Different situations, selected in a series of 125 mitotic and meiotic cytogenetic studies of Polyphaga beetle species, are reported and discussed, with the aim to improve our knowledge on the mechanisms of sex chromosome rearrangements, the relationships with nucleoli and the consequences on dosage compensation and chromosome segregation.

Nucleolus and chromosome relationships at pachynema in four Scarabaeoidea (Coleoptera) species with various combinations of NOR and sex chromosomes.

Nucleolus organizer regions (NORs) and nucleolus locations were studied after silver staining in spermatocytes at pachynema from four beetle species selected for their various combinations of sex chromosomes. Their karyotypic formulae were: 18,neoXY (Dorcus parallelipipedus); 25,X (Passalus unicornis) and 20,Xyp (Cetonia aurata and Protaecia (Potosia) opaca). NORs were located in the short arms of a unique acrocentric autosome pair in the first three and in intercalary position in a sub-metacentric autosome pair in the last species. Silver staining gave remarkably more consistent results in pachytene than in mitotic spreads, enabling the detection of both NORs and nucleoli, and also better results in embryo than in spermatogonial metaphases. At pachynema the NORs were elongated, roughly in proportion to the number of nucleoli, which always remained associated with NOR. Nucleoli were not recurrently associated with sex chromosomes, except in P. unicornis, at late pachynema. In C. aurata and P. opaca the sex body was recurrently associated with acrocentric short arms and metacentric telomeres, respectively. Even in these simple situations, with NORs located in a single autosome pair, the number of nucleoli and their relationships with sex chromosomes varied strongly from species to species. These variations appear to be largely determined by the chromosome rearrangements which have occurred during evolution, which makes extrapolations and generalizations quite hazardous. In D. parallelipipedus pachytene cells a quasi-systematic and transient fusion between the terminal heterochromatin of two sub-metacentrics was detected. Other chromosome bivalents could also be occasionally associated, but not the NOR carrier one. A strong enhancement of DAPI or quinacrine mustard staining was observed at the fusion point.

X-Y-autosome translocation, chromosome compaction, NOR expression and heterochromatin insulation in the Scarabaeid beetle Dynastes hercules hercules.

The karyotype of the giant beetle Dynastes hercules hercules is composed of only 16 autosomes and large sex chromosomes. Meiotic studies in the males showed that a large part of the sex chromosomes undergo synapsis at pachynema similarly to autosomes, demonstrating that both derived from an autosome-gonosome translocation. Therefore, karyotype formula is 18,neoXY. The heterochromatisation of the neoX short arm at pachynema indicates that it corresponds to the ancestral X. It carries the nucleolar organizer region (NOR) in its proximal part, which is undercondensed, especially in male mitotic and meiotic cells. In female mitotic cells, both NOR staining and undercondensation were more difficult to observe in the neoX short arms. In somatic interphase nuclei, NOR expression strongly varies with the sex. Two separated compact groups of silver dots were observed in female nuclei, while a single dispersed and large group of silver deposit exists in the males. Both the lower condensation and the higher NOR expression of the single neoX of the males, compared to each of the two neoXs of the females, is interpreted to be a consequence of dosage compensation, a mechanism not yet described in Coleoptera. In mammals as well as in Coleoptera, the carriers of gonosome-autosome translocations not exhibiting deleterious phenotypes show constitutive heterochromatin at the autosome-gonosome junction. Thus, heterochromatin may play an important universal role by clearly separating chromosome segments with different regulations of gene expression, such as inactivation or dosage compensation of the X chromosome on the one side and a conventional autosomal structure on the other side.

Potentiation of antitumour activity of docetaxel by combination with trastuzumab in a human prostate cancer xenograft model and underlying mechanisms.

Antitumour activity of docetaxel (Taxotere) in hormone-dependent (HD) and hormone-independent (HID) prostate cancer PAC120 xenograft model was previously reported, and its level was associated with HER2 protein expression. In the present study, we evaluate the antitumour effects of docetaxel combined with trastuzumab (Herceptin), an anti-HER2 antibody. Although trastuzumab alone had no effect on tumour growth, it potentiated the antitumour activity of docetaxel in HD tumours and more strongly in HID variants. Using the HID28 variant, we show that docetaxel treatment of tumour-bearing mice induces an increased HER2 mRNA expression of the tyrosine kinase receptor of 25-fold 24 h after docetaxel treatment, while HER2 protein and p-AKT decreased. This was followed by an increase of HER2 protein 3 days (two-fold) after docetaxel treatment and by a strong HER2 release in the serum of treated mice; expression of phospho-ERK, p27, BCL2 and HSP70 concomitantly increased. Similar molecular alterations were induced by docetaxel plus trastuzumab combination, except for that there was a transient and complete disappearance of AR and HSP90 proteins 24 h after treatment. We show that in addition to its known effects on tubulin and mitotic spindles, docetaxel induces complex signalisation pathway mechanisms in surviving cells, including HER2, which can be pharmacologically targeted. This study suggests that the docetaxel/trastuzumab combination may prove an effective therapeutic approach for HER2-expressing hormone-refractory prostate cancer.

High chromosomal polymorphism and heterozygosity in Cyclocephala tridentata from Guadeloupe: chromosome comparison with some other species of Dynastinae (Coleoptera: Scarabaeidae).

Very distinct karyotypes have been observed in two Cyclocephala species from Guadeloupe, considered as very close and possibly vicariant: C. insulicola with only metacentric and C. tridentata tridentata with many acrocentric autosomes. This prompted us to study the karyotype of a few other neotropical Dynastinae belonging to four of the eight existing tribes, to find out which one of these two species had the most divergent chromosomes from their ancestral condition. In the four additional species studied, i.e., Cyclocephalamaffafa, Strategus syphax, Ligyrus cuniculus and Megasoma actaeon, a karyotype composed of 20 chromosomes, including 18 meta- or submetacentric autosomes was found, as it was in C. insulicola. Thus, the karyotype of C. t. tridentata, in which most of the 18 autosomes were acrocentric, is apomorphic. In addition, it was highly polymorphic, with six different karyotypes observed among the ten specimens studied. All had one to four heterozygous chromosome pairs formed by one acrocentric and one submetacentric carrying a large juxta-centromeric heterochromatic component. This heterozygosity did not seem to impair either meiotic synapsis or chiasma formation and chromosome segregation. Such high rates of chromosome heterozygosity and polymorphism are infrequent and never described in beetles. This suggests that C. t. tridentata undergoes an active process of chromosome evolution. A possible relationship with insularity and/or pesticide exposure is briefly discussed.

Meiotic behaviour of a new complex X-Y-autosome translocation and amplified heterochromatin in Jumnos ruckeri (Saunders) (Coleoptera, Scarabaeidae, Cetoniinae).

Species belonging to the Cetoniinae subfamily studied so far possess 20 chromosomes, including a small X and a punctiform Y: 20,Xyp in the males. In a series of species from the Goliathini tribe under study we found a very unusual karyotype, with 12 autosomes and large sex chromosomes (14,neoXY) in Jumnos ruckieri from Thailand. Applying various techniques including pachytene bivalent spreading, we showed that 40% (mitotic and meiotic prophases) to 60% (metaphases) of the karyotype length was composed of heterochromatin. Both sex chromosomes were NOR carriers. At pachynema they underwent a complete synapsis of their distal regions, indicating their autosomal origin. At contrast, their very uneven central regions remained separated, but associated with nucleolus material. This association persisted until diakinesis, forming a pseudo-chiasma between the neoX and the neoY, which were always in end-to-end association. Compared to free autosomes the autosomal parts of the neo-sex chromosomes had a significant lack of interstitial chiasmata, indicating a possible lack of recombination at their proximal regions. As in the cases of X-autosome translocations in mammals, autosomal and gonosomal parts of the neo-sex chromosomes were insulated by heterochromatin, which may be a necessary condition to avoid deleterious position effects, whatever the mechanisms of gene dosage compensation.

Use of meiotic pachytene stage of spermatocytes for karyotypic studies in insects.

Coleopterans represent by far the largest animal group, with more than 300,000 identified species. Only little progress in their chromosome analysis has been accomplished during recent decades, compared with that made in vertebrate cytogenetics. Both the small size of their genome and the difficulty of obtaining mitotic cells with nice chromosomes have limited the application of conventional techniques, such as chromosome banding. A method for obtaining chromosome banding on well-spread bivalents from the pachytene stage of the meiotic prophase, the most frequent stage in young imagines, is described. It makes possible the identification of all bivalents and the establishment of the karyotype with greater ease and accuracy than with mitotic cells. In addition, it gives some insight into chromosome organization at a stage when autosomes are assumed to undergo an intense transcriptional activity. The results of the technique, which was successfully applied to many species, are described here in two of them, Cetonia aurata and Adesmia montana as examples.

Application of molecular cytogenetics for chromosomal evolution of the Lemuriformes (Prosimians).

R-banding chromosomal studies of 21 species of Lemuriformes allowed us to reconstruct the presumed ancestral karyotype of all the Lemuriformes except for Daubentoniidae and permitted the construction of their phylogenetic tree. Chromosome painting with fluorescently labeled heterologous DNA probes permitted comparative chromosome maps to be established. The Zoo-FISH method was used to reassess the karyotypes of 22 species or subspecies. While our results largely confirm the previous reconstruction of the ancestral karyotype, they resulted in a modification of the previously established phylogenetic tree. The Daubentoniidae emerged first followed by the divergence of the families Cheirogaleidae, Indriidae, Lepilemuridae and Lemuridae. Eight chromosome rearrangements occurred in all Lemuriformes except for Daubentoniidae in the common trunk. The present findings do not allow us to propose the occurrence of any rearrangement common to Daubentoniidae and other Lemuriformes, and probably other Prosimii. Conserved syntenies previously described in various mammalian orders were also conserved, while others were specific to the Lemuriformes.

Chromosome painting comparison of Leontopithecus chrysomelas (Callitrichine, Platyrrhini) with man and its phylogenetic position.

Using human probes of whole chromosomes, the homoeologies between human and Leontopithecus chrysomelas (Platyrrhini) karyotypes were established. Thirty-three conserved segments were observed between the two species. Intrachromosomal rearrangements between the two species were identified using hybridization of chromosome arm probes of human chromosomes 1 and 3. We also used chromosomal data to investigate phylogenetic relationships of Callitrichines. These data were encoded using Cebus capucinus , a species which kept fairly ancestral chromosomes, as reference. Two equi-parsimonious trees, including reversion or convergence events, were obtained. The monophyly of Callitrichines is confirmed. They share nine chromosomal rearrangements at least. The Cebuella-Callithrix group forms a clade sharing five rearrangements at least. According to the tree considered, the Tamarins, Leontopithecus and Saguinus share two chromosomal rearrangements restricted to these two taxa or none. Callimico accumulated seven chromosomal rearrangements unshared with other taxa, at least. To avoid convergence and reversion events, we propose the hypothesis of a network (or populational) evolution. Six chromosomal rearrangements would have occurred during the period of this network evolution. Finally, the karyotype of the last common ancestor to all Callitrichines has been reconstructed. It possessed 48 chromosomes.

DNA methylation in mouse gametogenesis.

DNA methylation is involved in many biological processes and is particularly important for both development and germ cell differentiation. Several waves of demethylation and de novo methylation occur during both male and female germ line development. This has been found at both the gene and all genome levels, but there is no demonstrated correlation between them. During the postnatal germ line development of spermatogenesis, we found very complex and drastic DNA methylation changes that we could correlate with chromatin structure changes. Thus, detailed studies focused on localization and expression pattern of the chromatin proteins involved in both DNA methylation, histone tails modification, condensin and cohesin complex formation, should help to gain insights into the mechanisms at the origin of the deep changes occurring during this particular period.

Highly conserved chromosomes in an Asian squirrel (Menetes berdmorei, Rodentia: Sciuridae) as demonstrated by ZOO-FISH with human probes.

The chromosomes of Menetes berdmorei (Rodentia, Sciuridae, Sciurinae) were studied by ZOO-FISH using whole human chromosome probes. All homoeologies between M. berdmorei and human chromosomes were determined, except for two small chromosome segments. Twelve human chromosomes are conserved in a unique block of synteny; ten are split into two and one into three blocks. Thus, a small number of interchromosomal rearrangements, about twenty, separates human from this squirrel karyotype. Homoeologies between human and the presumed ancestral chromosomes of Sciurinae could also be deduced, as well as those with the presumed ancestral chromosomes of eutherian mammals. Sciurinae chromosomes appear to be much closer to those of non-rodent mammals than those of Muridae and Cricetidae species studied so far. Thus, they provide an interesting tool to link the rodent genome to those of other mammals.

Reconstruction of the ancestral karyotype of eutherian mammals.

Applying the parsimony principle, i.e. that chromosomes identical in species belonging to different taxa were likely to be present in their common ancestor, the ancestral karyotype of eutherian mammals (about 100 million years old) was tentatively reconstructed. Comparing chromosome banding with all ZOO-FISH data from literature or studied by us, this reconstruction can be proposed with only limited uncertainties. This karyotype comprised 50 chromosomes of which 40-42 were acrocentrics. Ten ancestral pairs of chromosomes were homologous to a single human chromosome: 5, 6, 9, 11, 13, 17, 18, 20, X and Y (human nomenclature). Nine others were homologous to a part of a human chromosome: 1p + q (proximal), 1q, 2p + q (proximal), 2q, part of 7, 8q, 10p, 10q and 19p (human nomenclature). Finally, seven pairs of chromosomes, homologs to human chromosomes 3 + 21, 4 + 8p, part of 7 + 16p, part of 12 + part of 22 (twice), 14 + 15, 16q + 19q, formed syntenies disrupted in man.