Cytogenetic analysis of Epicauta atomaria (Meloidae) and Palembus dermestoides (Tenebrionidae) with Xyp sex determination system using standard staining, C-bands, NOR and synaptonemal complex microspreading techniques

The mitotic and meiotic chromosomes of the beetles Epicauta atomaria (Meloidae) and Palembus dermestoides (Tenebrionidae) were analysed using standard staining, C-banding and silver impregnation techniques. We determine the diploid and haploid chromosome numbers, the sex determination system and describe the chromosomal morphology, the C-banding pattern and the chromosome(s) bearing NORs (nucleolar organizer regions). Both species shown 2n = 20 chromosomes, the chromosomal meioformula 9 + Xyp, and regular chromosome segregation during anaphases I and II. The chromosomes of E. atomaria are basically metacentric or submetacentric and P. dermestoides chromosomes are submetacentric or subtelocentric. In both beetles the constitutive heterochromatin is located in the pericentromeric region in all autosomes and in the Xp chromosome; additional C-bands were observed in telomeric region of the short arm in some autosomes in P. dermestoides. The yp chromosome did not show typical C-bands in these species. As for the synaptonemal complex, the nucleolar material is associated to the 7th bivalent in E. atomaria and 3rd and 7th bivalents in P. dermestoides. Strong silver impregnated material was observed in association with Xyp in light and electron microscopy preparations in these species and this material was interpreted to be related to nucleolar material.

Cytogenetic And Morphologic Approaches Of Hybrids From Experimental Crosses Between Triatoma Lenti Sherlock & Serafim, 1967 And T. Sherlocki Papa Et Al., 2002 (hemiptera: Reduviidae).

The reproductive capacity between Triatoma lenti and Triatoma sherlocki was observed in order to verify the fertility and viability of the offspring. Cytogenetic, morphological and morphometric approaches were used to analyze the differences that were inherited. Experimental crosses were performed in both directions. The fertility rate of the eggs in crosses involving T. sherlocki females was 65% and 90% in F1 and F2 offspring, respectively. In reciprocal crosses, it was 7% and 25% in F1 and F2 offspring, respectively. The cytogenetic analyses of the male meiotic process of the hybrids were performed using lacto-acetic orcein, C-banding and Feulgen techniques. The male F1 offspring presented normal chromosome behavior, a finding that was similar to those reported in parental species. However, cytogenetic analysis of F2 offspring showed errors in chromosome pairing. This post-zygotic isolation, which prevents hybrids in nature, may represent the collapse of the hybrid. This phenomenon is due to a genetic dysregulation that occurs in the chromosomes of F1. The results were similar in the hybrids from both crosses. Morphological features, such as color and size of connexive and the presence of red-orange rings on the femora, were similar to T. sherlocki, while wins size was similar to T. lenti in F1 offspring. The eggshells showed characteristics that were similar to species of origin, whereas the median process of the pygophore resulted in intermediate characteristics in the F1 and a segregating pattern in F2 offspring. Geometric morphometric techniques used on the wings showed that both F1 and F2 offspring were similar to T. lenti. These studies on the reproductive capacity between T. lenti and T. sherlocki confirm that both species are evolutionarily closed; hence, they are included in the brasiliensis subcomplex. The extremely reduced fertility observed in the F2 hybrids confirmed the specific status of the species that were analyzed.

Comparative Cytogenetics Of Physalaemus albifrons And Physalaemus Cuvieri Species Groups (anura, leptodactylidae).

Recently, Physalaemus albifrons (Spix, 1824) was relocated from the Physalaemus cuvieri group to the same group as Physalaemus biligonigerus (Cope, 1861), Physalaemus marmoratus (Reinhardt & Lütken, 1862) and Physalaemus santafecinus Barrio, 1965. To contribute to the analysis of this proposition, we studied the karyotypes of Physalaemus albifrons, Physalaemus santafecinus and three species of the Physalaemus cuvieri group. The karyotype of Physalaemus santafecinus was found to be very similar to those of Physalaemus biligonigerus and Physalaemus marmoratus, which were previously described. A remarkable characteristic that these three species share is a conspicuous C-band that extends from the pericentromeric region almost to the telomere in the short arm of chromosome 3. This characteristic is not present in the Physalaemus albifrons karyotype and could be a synapomorphy of Physalaemus biligonigerus, Physalaemus marmoratus and Physalaemus santafecinus. The karyotype of Physalaemus santafecinus is also similar to those of Physalaemus marmoratus and Physalaemus biligonigerus owing to the presence of several terminal C-bands and the distal localization of the NOR in a small metacentric chromosome. In contrast, the Physalaemus albifrons karyotype has no terminal C-bands and its NOR is located interstitially in the long arm of submetacentric chromosome 8. The NOR-bearing chromosome of Physalaemus albifrons very closely resembles those found in Physalaemus albonotatus (Steindachner, 1864), Physalaemus cuqui Lobo, 1993 and some populations of Physalaemus cuvieri Fitzinger, 1826. Additionally, the Physalaemus albifrons karyotype has an interstitial C-band in chromosome 5 that has been exclusively observed in species of the Physalaemus cuvieri group. Therefore, we were not able to identify any chromosomal feature that supports the reallocation of Physalaemus albifrons.

Cytogenetic analysis of Astylus antis (Perty, 1830) (Coleoptera, Melyridae): Karyotype, heterochromatin and location of ribosomal genes

Cytogenetic analysis of Astylus antis using mitotic and meiotic cells was performed to characterize the haploid and diploid numbers, sex determination system, chromosome morphology, constitutive heterochromatin distribution pattern and chromosomes carrying nucleolus organizer regions (NORs). Analysis of spermatogonial metaphase cells revealed the diploid number 2n = 18, with mostly metacentric chromosomes. Metaphase I cells exhibited 2n = 8II+Xyp and a parachute configuration of the sex chromosomes. Spermatogonial metaphase cells submitted to C-banding showed the presence of small dots of constitutive heterochromatin in the centromeric regions of nearly all the autosomes and on the short arm of the X chromosome (Xp), as well as an additional band on one of the arms of pair 1. Mitotic cells submitted to double staining with base-specific fluorochromes (DAPI-CMA3) revealed no regions rich in A+T or G+C sequences. Analysis of spermatogonial mitotic cells after sequential Giemsa/AgNO3 staining did not reveal any specific mark on the chromosomes. Meiotic metaphase I cells stained with silver nitrate revealed a strong impregnation associated to the sex chromosomes, and in situ hybridization with an 18S rDNA probe showed ribosomal cistrons in an autosomal bivalent.

Karyotypic evolution trends in Rhamdia quelen (Siluriformes, Heptapteridae) with considerations about the origin and differentiation of its supernumerary chromosomes

Among catfish species of the genus Rhamdia reported for the Brazilian territory, R. quelen is the most widespread, being found in nearly all hydrographic basins of Brazil. Nowadays, R. quelen is a synonym for at least 47 other species in this genus, its taxonomic status still being controversial. The available cytogenetic reports show a wide variation in the karyotypic macrostructure, with the frequent presence of supernumerary chromosomes. The remarkable cytogenetic variability associated with taxonomic issues in this species indicates that R. quelen is actually a species complex. In order to carry out a wide comparative cytogenetic study in R. quelen from southern and southeastern Brazil and examine a species complex, we analyzed the chromosomes of 14 populations from the main hydrographic basins of these two regions. Using classic and molecular cytogenetic techniques, we found seven distinct karyotypic formulae, all bearing 2n = 58 chromosomes. Supernumerary chromosomes were present in most of the populations; their number, size and C-banding pattern allowed us to differentiate populations with similar karyotypic compositions. We examined patterns of chromosomal evolution as well as the probable mechanisms involved in the origin and morphological differentiation of their supernumerary chromosomes.

Mitotic and meiotic chromosomes of a southern Brazilian population of Boophilus microplus (Acari, Ixodidae)

Using conventional staining with acetic orcein and C-banding techniques it was investigated constitutive heterochromatin chromosomal polymorphisms and the mitotic and the meiotic behavior of male and female chromosomes of Boophilus microplus (Canestrini, 1887). Some differences were detected in the population of southern Brazil as compared to the data of other authors for populations in other latitudes. The differences being mainly concerned with the distribution of constitutive centromeric heterochromatin and variation in the length of heterochromatic blocks in the pericentromeric regions of some chromosome pairs.

Chromosomal characterization of Pseudonannolene strinatii (Spirostreptida, Pseudonannolenidae)

The chromosomes of the cave millipede Pseudonannolene strinatii Mauriès, 1974 were investigated. The diploid chromosome number was found to be 2n=16, XX/XY; the C-banding technique revealed a large amount of heterochromatin while the silver staining technique (Ag-NOR) evidenced the presence of heteromorphism of the NORs in some cells.

Chromosome studies of Brazilian vespertilionids Lasiurus cinereus and Lasiurus ega (Mammalia, Chiroptera)

Cytogenetical studies based on conventional coloration by Giemsa, C-banding and Ag-NOR were performed on 2 species of bats from the vespertilionid family: Lasiurus cinereus (Beauvois, 1796) and Lasiurus ega (Gervais, 1856). The 2n was 28 and FN was 48 in both species. The constitutive heterochromatin is located in centromeric regions in the two species and in the short arm of the subtelocentric X chromosome in L. ega. NORs were observed in the secondary constriction of the smaller autosome in both species.

A new species of Urostreptus (Diplopoda, Spirostreptidae): description and chromossome number

This work presents the description and chromosome number of Urostreptus atrobrunneus sp. nov. The genus until now had not been registered yet in the São Paulo State, Brazil. The meiotic analysis showed that the species presents 2n=24, XY. The C-banding revealed large blocks of constitutive heterochromatin and two heteromorphic chromosomal pairs, one of them corresponding to the sexual pair.

Comparative cytogenetics in different populations of the cavernicolous diplopod Pseudonannolene strinatii (Diplopoda, Pseudonannolenidae)

Different populations of Pseudonannolene strinatii Mauriès, 1974 collected from three caves in Iporanga, state of São Paulo, were cytogenetically compared using techniques of conventional coloration, C-banding and silver nitrate impregnation. Specimens were morphologically similar and small cytogenetic differences were observed between the populations with relation to the distribution of constitutive heterochromatin.

Cytochemical study of Rhodnius neglectus and Rhodnius prolixus salivary gland cells (Hemiptera, Triatominae)

Triatomines are hematophagous bugs of medical interest in South and Central America, where they may act as invertebrate hosts of the hemoflagellate protozoa Trypanosoma cruzi (the causative of Chagas’ disease) and Trypanosoma rangeli (Tejera, 1920). Triatomines of Rhodnius genus have salivary gland formed by two close and independent units: the principal and the accessory. This gland secretes saliva that abounds in substances that facilitate and permit feeding. Despite this importance, there are few reports on its cytochemistry. In purpose of amplifying this understanding, in this work it was investigated the nuclear structures (chromatin and nucleolar corpuscles) of salivary gland cells of Rhodnius neglectus (Lent, 1954) and Rhodnius prolixus (Stål, 1859). The salivary glands were removed from adult insects, fixed and submitted to different cytochemical methods: lacto-acetic orcein, silver ion impregnation, Feulgen reaction, Toluidine Blue, Variant method of critical electrolyte concentration and C-banding. The results evidenced predominance of binucleated cells, with bulky and polyploid nucleus, decondensed chromatin and a large nucleolar area. In addition, cytoplasmic metachromasy and a clear association between nucleolar and heterochromatic corpuscles were observed. Such characteristics were associated with intense synthesis activity to produce saliva. Besides, the heterochromatic corpuscles observed with C Banding permitted the differentiation of sexes and species.

Cytogenetics as a tool for Triatomine species distinction (Hemiptera-Reduviidae)

Several cytogenetic traits were tested a species diagnostic characters on five triatomine species: Rhodnius pictipes, R. nasutus, R. robustus, Triatoma matogrossensis and T. pseudomaculata. Four of them are described for the first time. The detailed analysis of the meiotic process and the application of C-banding allowed us to identify seven cytogenetic characters wich result useful to characterize and differentiate triatomine species.

Location of ribosomal genes in the chromosomes of Anopheles darlingi and Anopheles nuneztovari (Diptera, Culicidae) from the Brazilian Amazon

Fluorescence in situ hybridization of Anopheles darlingi and A. nuneztovari demonstrated nucleolar organizer region activity at the end of the fourth larval instar, when the nucleolar organizer regions underwent gradual condensation. The heteromorphic sex chromosomes showed intraindividual size variation in the rDNA blocks located in the pericentromeric region and this coincided with the location of constitutive heterochromatin (C-banding).

Chromosome variability in the Chagas disease vector Rhodnius pallescens (Hemiptera, Reduviidae, Rhodniini)

Rhodnius pallescens is the main vector of Trypanosoma cruzi in Panama and one of the most relevant secondary vectors in Colombia. Despite the importance of this species, there is limited knowledge about the genetic variability along its geographical distribution. In order to evaluate the degree of karyotype variability we analyzed the meiotic behavior and banding pattern of the chromosomes of 112 males of R. pallescens coming from different regions of Colombia and Panama. Using the C-banding technique we identified two chromosomal patterns or cytotypes characterized by differences in the amount, size and distribution of constitutive heterochromatic regions in the chromosome complement (2n = 20 autosomes plus XY in males). The individuals can be easily classified in each cytotype by the analysis of the chromosomes during first meiotic prophase. The frequencies of the cytotypes are variable according to the geographic origin of the populations. This chromosomal divergence together with morphological data supports the existence of three genetically different populations of R. pallescens and provides new information to understand the distribution dynamics of this species.

Recuperation and improvement of chromosome preparations stained with silver nitrate

Both periodic acid and photographic reagents have been used to remove the silver nitrate residues from cytological preparations. We used potassium ferricyanide to remove AgNO3 salts in cases of excessive chromosome impregnation. This method produced partial decolorization, with contrast enhancement. Counterstaining with Giemsa also promoted a better contrast between chromosome arms and NORs, which were preserved and retained a dark color. Application of this procedure to chromosome preparations treated sequentially for CBG banding/AgNO3 staining promoted complete decolorization of C+ regions, leading to "reverse" C-banding, generally with high contrast.