Pycnotic cycle of the sex chromosome of Pyrgomorpha conica (Orthoptera) and development of spermiogenesis.

We have analyzed the anomalous pycnotic cycle of the X sex chromosome of the grasshopper Pyrgomorpha conica throughout both meiotic divisions and its possible influence on spermiogenesis. During diplotene the sex chromosome shows two differentiated pycnotic regions: (i) the centromeric region, which is negatively heteropycnotic, and (ii) the noncentromeric region, which shows alternating negatively and positively heteropycnotic zones in all standard individuals. The variation in size and location of the negative heteropycnotic zones, their smooth appearance, and their lack of effect on spermiogenesis lead us to suggest that condensation differences and not euchromatinization are responsible for their presence. In two individuals the sex chromosome appeared partially isopycnotic at metaphase I, and high levels of abnormal spermatids (macrospermatids and microspermatids) were found. We suggest that the possible activity of this chromosome during the second meiotic division may promote the disruption of spermiogenesis by affecting the mechanism that maintains intercellular bridges between normal spermatids.     

Supernumerary segments in five species of grasshoppers (Orthoptera: Acridoidea)

Supernumerary segments have been observed in five species of grasshoppers: Calliptamus barbarus, Oedipoda fuscocincta, Acrotylus insubricus, Omocestus raymondi and Chorthippus ariasi. In four cases they are located in the S-chromosomes, but in A. insubricus they are carried by the megameric pair (M₉). In C. barbarus and O. fuscocincta we have observed non-homologous association during diplotene between the extra segments and the X-chromosome. The supernumerary segments of these two species are distally located. However, in anaphase-I the unequal bivalents divide reductionally in 30% and 20% of the cells, respectively. Finally, the supernumerary segments of C. barbarus do not produce any effect on mean chiasma frequency, but they decrease significantly the between-cell variance of chiasmata in males carrying them.     

Interstitial telomeric sequence blocks in constitutive pericentromeric heterochromatin from Pyrgomorpha conica (Orthoptera) are enriched in constitutive alkali-labile sites

The long interstitial telomeric repeat sequence (ITRS) blocks located in the pericentromeric chromosomal regions of most of Chinese hamster chromosomes behave as hot spots for spontaneous and induced chromosome breakage and recombination. The DBD-FISH (DNA breakage detection-fluorescence in situ hybridization) procedure demonstrated that these ITRS are extremely sensitive to alkaline unwinding, being enriched in constitutive alkali-labile sites (ALS). To determine whether this chromatin modification occurs in other genomes with large ITRS that are not phylogenetically related to mammalian species, the grasshopper Pyrgomorpha conica was analyzed. We chose this species because, with conventional FISH, their chromosomes yield extremely small telomeric signals when probed with the (TTAGG)n polynucleotide, but large ITRS blocks as part of their pericentromeric constitutive heterochromatin. A high density of constitutive ALS was evidenced in the ITRS when intact meiotic cells or somatic cells were subjected to the DBD-FISH technique and probed with the specific telomeric DNA. DBD-FISH with simultaneous hybridization using telomeric and whole genome DNA probes showed that the ITRS tend to colocalize with areas of stronger signal from the whole genome probe. Nevertheless, the signal from the whole genome was more widespread than that from the ITRS, thus providing evidence that a high frequency of constitutive ALS was present in more than one DNA sequence type. Furthermore, stretched DNA fibers processed with DBD-FISH, revealed a distribution of telomeric sequences alternating interspersed with other possible highly repetitive DNA sequences. The abundance of ALS varied from one meiotic stage to another. Interestingly, most of the breakage and meiotic recombination in males takes place close to the constitutive heterochromatin, particularly enriched in ALS. These results provide further evidence of a particular, and possible universal, chromatin structure enriched in constitutive ALS at constitutive heterochromatic regions.     

Sex chromosomes and associated rDNA form a heterochromatic network in the polytene nuclei of Bactrocera oleae (Diptera: Tephritidae)

The olive fruit fly, Bactrocera oleae, has a diploid set of 2n?=?12 chromosomes including a pair of sex chromosomes, XX in females and XY in males, but polytene nuclei show only five polytene chromosomes, obviously formed by five autosome pairs. Here we examined the fate of the sex chromosomes in the polytene complements of this species using fluorescence in situ hybridization (FISH) with the X and Y chromosome-derived probes, prepared by laser microdissection of the respective chromosomes from mitotic metaphases. Specificity of the probes was verified by FISH in preparations of mitotic chromosomes. In polytene nuclei, both probes hybridized strongly to a granular heterochromatic network, indicating thus underreplication of the sex chromosomes. The X chromosome probe (in both female and male nuclei) highlighted most of the granular mass, whereas the Y chromosome probe (in male nuclei) identified a small compact body of this heterochromatic network. Additional hybridization signals of the X probe were observed in the centromeric region of polytene chromosome II and in the telomeres of six polytene arms. We also examined distribution of the major ribosomal DNA (rDNA) using FISH with an 18S rDNA probe in both mitotic and polytene chromosome complements of B. oleae. In mitotic metaphases, the probe hybridized exclusively to the sex chromosomes. The probe signals localized a discrete rDNA site at the end of the short arm of the X chromosome, whereas they appeared dispersed over the entire dot-like Y chromosome. In polytene nuclei, the rDNA was found associated with the heterochromatic network representing the sex chromosomes. Only in nuclei with preserved nucleolar structure, the probe signals were scattered in the restricted area of the nucleolus. Thus, our study clearly shows that the granular heterochromatic network of polytene nuclei in B. oleae is formed by the underreplicated sex chromosomes and associated rDNA.     

Two repeated DNA sequences from the heterochromatic regions of rye (Secale cereale) chromosomes

Rye DNA sequences renaturing with a C₀t <0.02 mol·sec/l, are largely undigested by the restriction enzyme HindIII. These HindIII-spared sequences are mostly located in telomeric heterochromatin. When digested with EcoRI* and cloned into the EcoRI site of pBR 325, these sequences yielded clones of two classes when hybridized to a probe of rapidly renaturing DNA. One class contains a DNA sequence which is a major constituent of the telomeric heterochromatic blocks, while the other is a minor component of the highly repeated DNA of the genome. The major component was sequenced, its chromosomal distribution mapped using wheat-rye addition lines and its distribution in meiotic prophase nuclei determined. The minor component is present in significant amounts in wheat as well as in rye and is localized at the terminal heterochromatic regions of three rye chromosomes but not in the major blocks of heterochromatin.     

Morphology,development and reproduction of Pyrgomorpha vignaudii (Orthoptera: Pyrgomorphidae)

Pyrgomorpha vignaudii (Guérin‐Méneville, 1849) is a pest of a wide variety of crop plants in Africa. To facilitate the search for a sustainable strategy against this pest, we have studied the post‐embryonic development, morphology and reproduction on Manihot esculenta in the laboratory. Five‐hundred and two larvae at the first stage of development obtained in the laboratory were individually reared in cages. Post‐embryonic development passed through seven stages. The total number of days spent for larval development varied from 77 to 108. The mean duration of development of stages 1, 2, 3, 4, 5 and 6 larvae were, respectively, 16.48 ± 0.43, 14.40 ± 0.55, 13.70 ± 0.61, 15.07 ± 0.84, 15.21 ± 1.31 and 16.21 ± 1.27 days. After the final molt, the females of P. vignaudii take an average of 12.7 ± 1.04 days before the first mating. The time between the first mating and first oviposition ranged from 14 to 34 days (averagely 25.2 ± 4.62 days). The females realized one to nine ovipositions during their lives. The number of eggs per egg‐pods (ootheca) varied from 16 to 93 with a mean of 45.31 ± 3.51. Our results provide valuable information for the search for a control strategy against P. vignaudii.     

Non-random segregation during anaphase II in an individual of Arcyptera tornosi (Bol.) (Orthoptera) heterozygous for three supernumerary heterochromatic segments

An individual of Arcyptera tornosi heterozygous for distal heterochromatic segments affecting M₆, S₁₀ and S₁₁ chromosomes has been analyzed during all the meiotic stages in order to establish the pattern of meiotic segregation in anaphase I and II. S-bivalents invariably show an equational separation during anaphase I and the anaphase II separation is non-random, both chromatids with heterochromatic segments often segregating to the same pole. Differences are significant if compared with the expected segregation. Some aspects of this particular chromosome behaviour are briefly discussed.     

Prevalence of B chromosomes in Orthoptera is associated with shape and number of A chromosomes

We analyze the prevalence of B chromosomes in 1,601 species of orthopteran insects where chromosome number and shape are known. B chromosomes have been reported in 191 of these species. Bs are not uniformly distributed among orthopteran superfamilies, with evident hotspots in the Pyrgomorphoidea (32.3% of species carrying Bs), Grylloidea (14.9%), Acridoidea (14.6%) and Tetrigoidea (14.3%). As expected under the theory of centromeric drive, we found a correlation between B chromosome presence and A chromosome shape-Bs are more frequent in karyotypes with more acrocentric A chromosomes. We also found that Bs are less common in species with high chromosome numbers and appear to be most common at the modal chromosome number (2n = 24). Study effort, measured for each genus, was not associated with B prevalence, A chromosome shape or A chromosome number. Our results thus provide support for centromeric drive as an important and prevalent force in the karyotypic evolution of Orthoptera, just as it appears to be in mammals. We suggest that centromeric drive may provide a mechanistic explanation for White's principle of karyotypic orthoselection.     

Localization of specific rDNA spacer sequences to the mouse L-cell nucleolar matrix.

Mouse L-cell nucleoli were isolated from sonicated nuclei by centrifugation and extensively treated with pancreatic DNase or micrococcal nuclease to obtain "core nucleoli." Core nucleoli still contained the precursors to rRNA and about 1% of the total nuclear DNA, which remained tightly bound even after the removal of some chromatin proteins with 2 M NaCl. The core nucleolar DNA electrophoresed in a series of discrete bands, 20 to about 200 base pairs in length. Hybridization tests with specific DNA probes showed that the DNA was devoid of sequences complementary to mouse satellite, mouse Alu-like, and 5S RNA sequences. It also lacked sequences coding for cytoplasmic rRNA species, since it did not hybridize to the 18S to 28S portion of rDNA in Northern blot analyses and none of it was protected by hybridization to a 100-fold excess of total cytoplasmic RNA in S1 nuclease assays. However, the core nucleolar DNA did hybridize to nontranscribed and external transcribed spacer rDNA sequences. We infer that specific portions of rDNA are protected from DNase action by a tight association with nucleolar structural proteins.     

B chromosomes in Nierembergia aristata (Solanaceae): nucleolar activity and competition with the A chromosomes

B chromosomes are additional dispensable chromosomes that may be present in some individuals, populations, or species, which have probably arisen from the A chromosomes but follow their own evolutionary pathway. Supposedly, B chromosomes do not contain major genes except for ribosomal DNA (rDNA) sequences that have been mapped on the supernumerary chromosomes of many plants and animals. This paper is a new report of B chromosome occurrence in plants. B chromosomes with nucleolar organizing regions (NORs) were found in a diploid sample of Nierembergiaaristata D. Don (sub nom. N. stricta Miers) (2n = 2x = 16). This is an extreme case in which B chromosomes possess not only strong nucleolar activity, as revealed by conventional staining methods, AgNOR and fluorescence banding, and fluorescent in situ hybridization (FISH), but also show nucleolar competition with the A chromosomes. The observed phenomenon could be analogous to the nucleolar dominance or 'differential amphiplasty' phenomenon that occurs in interspecific hybrids.     

The 5S rDNA related repetitive sequences in the sex chromosomes of the spiny eel (Mastacembelus aculeatus)

The karyotype of the spiny eel (Mastacembelus aculeatus) has highly evolved heteromorphic sex chromosomes. X and Y chromosomes differ from each other in the distribution of heterochromatin blocks. To characterize the repetitive sequences in these heterochromatic regions, we microdissected the X chromosome, constructed an X chromosome library, amplified the genomic DNA using PCR and isolated a repetitive sequence DNA family by screening the library. All family members were clusters of two simple repetitive monomers, MaSRS1 and MaSRS2. We detected a conserved 5S rDNA gene sequence within monomer MaSRS2; thus, tandem-arranged MaSRS1s and MaSRS2s may co-compose 5S rDNA multigenes and NTSs in M. aculeatus. FISH analysis revealed that MaSRS1 and MaSRS2were the main components of the heterochromatic regions of the X and Y chromosomes. This finding contributes additional data about differentiation of heteromorphic sex chromosomes in lower vertebrates.     

The diminution of heterochromatic chromosomal segments in Cyclops (Crustacea,Copepoda)

The chromosomes of Cyclops divulsus, C. furcifer, and C. strenuus, like those of several other Copepods, undergo a striking diminution of chromatin early in embryogenesis. The process is restricted to the presumptive soma cells and occurs at the 5th cleavage in C. divulsus, at the 6th and 7th in C. furcifer, and at the 4th in C. strenuus. The eliminated chromatin derives from the excision of heterochromatic chromosome segments (H-segments). Their chromosomal location is different in the three investigated species: Whereas in C. divulsus and C. furcifer the H-segments form large blocks — exclusively terminal in the former and terminal as well as kinetochoric in the latter — the germ line heterochromatin in C. strenuus is scattered all along the chromosomes. Extensive polymorphism exists with respect to the length of the terminal H-segments in C. furcifer, and with respect to the overall content of heterochromatin in the chromosomes of C. strenuus. In a local race of C. strenuus an extreme form of dimorphism has been found which is sex limited: females as a rule are heterozygous for an entire set of large (heterochromatin-rich), and a second set of small chromosomes in their germ line. Males are homozygous for the large set. In the first three cleavage divisions the H-polymorphism is solely expressed through differences of chromosome length. Following diminution the differences between homologous have disappeared. Feulgen cytophotometry demonstrates that in the three species the 1C DNA value for the germ line, as measured in sperm, is about twice that measured in somatic mitoses (germ line/soma C-values in picograms of DNA: C. strenuus 2.2/0.9, C. furcifer 2.9/1.44, C. divulsus 3.1/1.8). — The data imply that chromatin diminution is based on a mechanism which allows specific DNA segments, regardless of their location and size, to be cut out from the chromosomes without affecting the structural continuity of the remaining DNA. This mechanism may be analogous to that of prokaryotic DNA excision.     

Supernumerary chromosomes,Robertsonian rearrangement and variability of the sex chromosomes inNectomys squamipes (Cricetidae,Rodentia)

Chromosomes were analyzed in 91 specimens ofNectomys squamipes, collected in three distinct geographic regions of Brazil. Chromosomal polymorphism due to supernumerary chromosomes, Robertsonian rearrangement and variation of sex chromosomes is reported. Samples collected in the northeastern region had 2n=52, FN=52; 2n=53, FN=54; 2n=54, FN=56; 2n=55, FN=56; 2n=56, FN=56; 2n=57, FN=57. The specimens from the southeastern and southern regions showed 2n=55, FN=56; 2n=56, FN=56; 2n=57, FN=58; 2n=58, FN=60; 2n=59, FN=62. Seven different types of supernumeraries were observed. These vary in size, morphology and C-banding characteristics.The Brazilian populations ofNectomys squamipes are composed of at least two basic entities, one with 2n=52 and its increasing series up to 2n=55, the other with 2n=56, increasing to 2n=59. Both entities are characterized by variation from 0 to 3 supernumeraries. The X and Y chromosomes present polymorphisms in terms of the size, shape as well as the distribution of constitutive heterochromatin.This research was supported by CNPq-PIG (Projeto Integrado de Genética) and is part of the Rodent Cytogenetic Colaborative Study, developed by different Laboratories in Brazil.     

Differential Giemsa staining of heterochromatic B-chromosomes in Myrmeleotettix maculatus (Thunb.) (Orthoptera: Acrididae)

A modified Giemsa staining technique has been used to investigate the karyotype of the grasshopper Myrmeleotettix maculatus, since this stain appears to be diagnostic for certain repetitive DNAs. The centromeric regions are densely heterochromatic, and further heterochromatic bands occur on the X-chromosome and on both arms of the B-chromosome. The significance of these results is discussed in relation to centromeric structure and B-chromosome structure in this insect, and a possible origin of the B-chromosome is suggested.