Cytogenetic study of holocentric chromosomes in three species of triatomines (Heteroptera, Reduvidae)

In the present work, spermatogenesis was analyzed in 3 species of the genus Triatoma (T. platensis, T. proctata, T. tibiamaculata). Lacto-acetic orcein staining was used in order to investigate chromosomal meiotic behavior of these species. It allowed the identification of the T. tibiamaculata karyotype (20, X 1X 2Y), the observation that in T. protacta doesn't occur late migration of sexual chromosomes and corroborated knowledgments about holocentric chromosome nature.

Description of the nucleolar activity and karyotype in germinative cell lines of Rhodnius domesticus (Triatominae, Heteroptera)

The purpose of this work was to study the karyotype, spermatogenesis and nucleolar activity at meiosis, in the species Rhodnius domesticus (Heteroptera, Triatominae). The testicular tubules were cytologically prepared by the conventional method of cell crushing and subsequent application of cytogenetic staining techniques with lacto-acetic orcein and silver-ion impregnation. The species under study presented karyotype 2n= 20A+XY, the modal number of the subfamily Triatominae. The chromosomes presented no primary constriction and were therefore characterized as holocentric. It was observed that the sex chromosomes sometimes were located at the periphery, close to the ring formed by autosomes, at first meiotic division. At metaphases II, sex chromosomes were positioned in the center of the autosomal ring, thus evidencing a postreductional behavior. These same chromosomes showed late migration at anaphases and were clearly impregnated with silver-ions, suggesting they bore Nucleolar Organizer Regions. Dispersed nucleolar corpuscles in cytoplasm until telophase II and small dots in spermatids strongly impregnated with silver, could be seen. Thus, it may be inferred that, in triatomines, the nucleolus does not completely disappear but remains in the form of small corpuscles that have a role in cell differentiation.

Cytogenetic study on three species of the genus Triatoma (Heteroptera: Reduviidae) with emphasis on nucleolar organizer regions

The use of banding techniques allows the recognition of chromosomal pairs and karyotypical arrangements. However, its application in Heteroptera holocentric chromosomes is limited. Thus, little is known about their structure, specially their Nucleolar Organizer Regions (NORs). A comparative analysis of the nucleolar characteristics present during spermatogenesis in Triatoma platensis, Triatoma protacta and Triatoma tibiamaculata seems to indicate that in this group of insects nucleolar fragmentation occurs after prophase I. The study of chromosomal structure of these triatomines indicates that NORs are located at some telomeric and interstitial autosome regions and at sexual chromosomes (X/X1X2).

Localization of rDNA sites in holocentric chromosomes of three species of triatomines (Heteroptera, Triatominae).

Chromatin organization in the holocentric chromosomes of three triatomines species was cytologically studied by fluorescent in situ hybridization with a 45S rDNA probe of Drosophila melanogaster to localize ribosomal genes. In Triatoma tibiamaculata, metaphases I showed telomeric highlights in a single, larger bivalent. In T. protacta, hybridization was detected in one of the telomeres of an autosomal chromosome. In T. platensis, there were highlights in a single, smaller chromosome (X chromosome). The results obtained did not agree with the expected localization of rDNA genes in the sex chromosomes of triatomines, as demonstrated by silver impregnation, and suggest that the chromosome reorganization that occurred in this group during evolution may be a more important mechanism involved in rDNA distribution.

Cytogenetical aspects of testicular cells in economically important species of coreidae family (Heteroptera)

Some cytogenetical aspects of spermatozoa formation were studied in 9 Coreidae Brazilian species: Anasa bellator, Athaumastus haematicus, Chariesterus armatus, Dallacoris obscura, Dallacoris pictus, Leptoglossus gonagra, Leptoglossus zonatus, Sphictyrtus fasciatus, and Zicca annulata. Similarly to the other species described to date, all the species studied herein showed cystic spermatogenesis, a reddish membrane covering the testes, a X0 sex determining system, a pair of m-chromosomes, intersticial chiasmata in most autosomes, and autosomes dividing reductionally at first meiotic division and equationally in the second 1 while sex chromosomes, divide equationally and reductionally at first and second meiotic division, respectively. In addition, it was observed that the sex chromosome is heteropycnotic at prophase and that heteropycnotic chromosomal material is found in the nuclei at spermiogenesis. In the species studied, the diploid chromosome number ranged from 19 to 25. It was 19 in S. fasciatus (16A+2m+X0); 21 in A. bellator, A. haematicus, D. obscura, D. pictus, L. gonagra, and L. zonatus (18A+2m+X0); 23 in Z. annulata (20A+2m+X0); and 25 in C. armatus (22A+2m+X0). © 2007 The Japan Mendel Society.

Study of chromosomal and nucleolar aspects in testes of Nysius californicus (Heteroptera: Lygaeidae)

In Nysius californicus (family Lygaeidae, subfamily Orsillinae), a pest commonly known as the seed bug, the chromosome complement is 2n = 16 (12A + 2m + XY), testes are formed by seven seminiferous tubules covered by an orange-colored membrane, and spermatogenesis is cystic. At prophase, sex chromosomes are heteropycnotic and autosomes usually show a chiasma. At metaphase, sex chromosomes along with microchromosomes may be seen located at the center of a ring formed by the remaining autosomes. A characteristic specific of N. californicus was the presence of nucleolar material observed from the cystic cell to the completely differentiated spermatozoon. Variations in size, shape and location of the nucleolar material occur during this process, denoting a variable degree of activity in the different stages. ©FUNPEC-RP.

Differential characterization of holocentric chromosomes in triatomines (Heteroptera, Triatominae) using different staining techniques and fluorescent in situ hybridization

A comparative study of holocentric chromosomes in the triatomine species Panstrongylus megistus, Rhodnius pallescens and Triatoma infestans was carried out in order to characterize heterochromatin, rDNA active sites and nucleolar proteins. Cytological preparations of seminiferous tubules were stained by silver impregnation, C banding, fluorochromes CMA 3/DA and DAPI/DA, and fluorescent in situ hybridization (FISH) with Drosophila melanogaster 28S rDNA probe. Our results showed interesting aspects of the organization of chromatin and chromosomes in the meiotic cells of these insects. In R. pallescens, sex chromosomes (X, Y) were distinct from autosomes, when submitted to silver impregnation, C banding, CMA 3 staining, and FISH, confirming that these chromosomes bear nucleolar organizer regions (NORs). In P. megistus, two of the three sex chromosomes were CMA 3/DAPI-; at early meiotic prophase and at diakinesis, silver impregnation corresponded with FISH signals, indicating that in this species, two chromosomes (probably a sex chromosome and an autosome) bear NORs. In T. infestans, silver nitrate and FISH also stained corresponding areas on meiotic chromosomes. Our data suggest that in triatomines, in general, the number and location of NORs are species-specific. These regions may be considered important chromosome markers for comparative studies to improve the understanding of evolutionary mechanisms in these hematophagous insects. ©FUNPEC-RP.

Pattern of silver nitrate-staining during meiosis and spermiogenesis in testicular lobes of Antiteuchus tripterus (Heteroptera: Pentatomidae)

The pattern of silver nitrate (Ag)-staining differed among testicular lobes of Antiteuchus tripterus. In general, these differences are in regard to the number, size, shape, coloring intensity, and location of the stained bodies or masses, observed during meiosis and spermiogenesis. These characteristics were similar in lobes 1-3. Lobes 4-6, however, differed from each other and from lobes 1-3 as well. Because the Ag-staining method is specific for nucleolar organizing regions and nucleolar material, the observations in meiosis of lobes 1-3 suggested the presence of a single pair of nucleolar organizing region-bearing chromosomes in A. tripterus, as previously found in other Pentatomidae species. In general, the amount of Ag-stained material seen in meiosis of the testicular lobes 1-3 of A. tripterus is smaller than in the other lobes. The differences among lobes observed during spermiogenesis included a striking variation in morphology of the Ag-stained material found in the head and tail of the spermatids. Given that the key role of the nucleolar material is to participate in protein synthesis, interlobular variations seem to be related to the different functions attributed to each lobe (reproduction to lobes 1-3 and basically nutrition to lobes 4-6). To our knowledge, this is the first time that the nucleolar material was studied in each testicular lobe during spermatogenesis. The present observations encourage further studies since, in addition to being of basic biological interest, several Pentatomidae species are agricultural pests and added knowledge of their biology, mainly in reproduction, may be important for the development of control strategies. ©FUNPEC-RP.

Cytogenetic analysis in the spermatogenesis of Triatoma melanosoma (Reduviidae; Heteroptera)

Triatomines are of great concern in public health because they are vectors of Chagas' disease. This study presents an analysis of the species Triatoma melanosoma. The cytogenetic characteristics of triatomines include holocentric chromosomes, post-reductional meiosis in the sex chromosomes and nucleolar fragmentation in the meiotic cycle. The methodology utilized consisted of the techniques of lacto-acetic orcein staining and silver ion impregnation. The organs analyzed were adult testicles. The results enabled to classify the chromosomes by number and size, being three large, eight medium and one small heterochromosome. The three largest chromosomes and the heterochromosomes showed heteropyknotic chromatin in meiosis. The heterochromosomes in 8.05% of the cells in metaphase I behaved as pseudobivalents, contrasting with 91.95% of the cells with individualized sex chromosomes, confirming the achiasmatic nature of these chromosomes. However, the pseudobivalents occurred prominently in metaphase II (78.38%), this fact probably is related to the post-reductional nature of the sex chromosomes. The nucleolus in T. melanosoma persisted until the diplotene phase after which it began to fragment. Nucleolar corpuscles were observed in metaphases I and II and during anaphases I and II, these characteristics being related to the phenomenon of nucleolar persistence. In the initial spermatids, peripheral silver ion impregnation occurred, which could be analogous to the pre-nucleolar corpuscles observed after fragmentation. Thus, this study extends our knowledge of the characteristics of triatomines, in particular, heteropyknotic degree, kinetic activity, formation of sex chromosome achiasmatic pseudobivalency, confirmation of the fragmentation phenomenon, and post-meiotic nucleolar reactivation. ©FUNPEC-RP.

Heteropicnotic chromatin and nucleolar activity in meiosis and spermiogenesis of Limnogonus aduncus (Heteroptera, Gerridae): a stained nucleolar organizing region that can serve as a model for studying chromosome behavior.

Males of Limnogonus aduncus were found to have the sex chromosome system X0 and chromosome number 2n = 23 (22A + X0). Testis cells were stained with lacto-acetic orcein and silver nitrate so that changes in the morphology and degree of staining of the heteropicnotic chromatin and the nucleolar material could be observed during meiosis and spermiogenesis. These structures share the same nuclear position and could be seen until almost the end of spermiogenesis. A chromosome region stained with silver nitrate was indicative of a nucleolar organizing region (NOR), which is rarely detected in Heteroptera with this technique. The NOR is located at one end of a single member of an autosome pair. The finding of this stained region enabled us to observe that the telomeric association of sister chromatids that characterizes the Heteroptera does not include the chromosome ends, where NORs are located; we also observed in anaphase that the chromosome end through which it is pulled to the pole is the one containing the NOR. Another observation was that the single nucleolar body present in the cells at anaphase never goes to the cell pole that does not receive the NOR. We conclude that L. aduncus is a good model for cytogenetic studies involving nucleolar activity and also may be useful for studying the mechanisms of activation and inactivation of kinetic activity at the chromosome ends. Although the chromosomes of Heteroptera are known to be holocentric, whether kinetic activity is restricted to one or involves both chromosome ends is still not well understood.

Bites caused by Giant Water Bugs belonging to belostomatidae family (Hemiptera, Heteroptera) in humans: A Report of Seven cases

We report 7 cases of patients bitten by giant water bugs, large predatory insects belonging to the Belostomatidae family (Hemiptera, Heteroptera). These insects have toxic saliva capable of provoking intense pain and paralysis in vertebrates. Victims experienced intense, excruciating pain and 1 manifested hypoesthesia in the forearm. Bites by Belostomatidae are often reported by clinicians working in areas where these insects live, but there are no detailed case reports in the medical literature. There are no specific treatment modalities known to be effective, making prevention an important strategy. © 2010 Wilderness Medical Society.

Spermatogenesis and karyotypes of three species of water striders (Gerridae, Heteroptera).

Although they are of economic importance, there have been few cytogenetic studies of the Gerridae (Heteroptera) in Brazil. We examined spermatogenesis (meiosis and spermiogenesis) and nucleolar behavior in three species of the family Gerridae. Brachymetra albinerva and Halobatopsis platensis were found to have a chromosome complement of 2n = 25 (24A + X0) and Cylindrostethus palmaris 2n = 29 (28A + X0) chromosomes. Fifteen individuals of these species were collected from the reservoir of São José do Rio Preto, SP, using screens and were transported in pots containing water to the laboratory, where cytogenetic preparations were made. The polyploidy nuclei are formed by several heteropyknotic regions; cells in meiotic prophase have a heteropyknotic region that is probably the sex chromosome, and the chromosomes from chiasmata. The spermatids are rounded and have a heteropyknotic region at the periphery of the nucleus; the sperm head is small, with a long tail. Silver impregnation of meiotic cells showed one or more disorganized bodies around the perichromosomal sheath. The round spermatids had two bodies next to each other, but these were elongated; one of the bodies remained in the head and the other migrated to the initial part of the tail at the end of spermagenesis, when the staining was no longer evident. The meiotic cells appear during spermatogenesis and have very similar silver-impregnation patterns in different species of Heteroptera.

Comparative study of spermatogenesis and nucleolar behavior in testicular lobes of Euschistus heros (Heteroptera: Pentatomidae)

In some testicular lobes of the Pentatomidae there may be occurrence of atypical spermatogenesis or polymegaly, leading to the production of nonfertile sperm. The comparative analysis of spermatogenesis and nucleolar behavior in testicular lobes of Euschistus heros showed cells with polymegaly in lobes 4 and 6. Generally, when these lobes are present in the same individual, there is also the formation of atypical cells in the flanking lobe. Such characteristic was not seen in E. heros. However, differences regarding the concentration of heteropyknotic chromatin and silver-positive bodies in this lobe deserve attention. This study explored the literature and demonstrated the prevalence of some lobes in the formation of differentiated cells. It was also found in the literature that there is an association of the chromocenter with the nucleolus in several species of Pentatomidae, but in E. heros this association does not appear to occur. © 2010 Hederson Vinicius de Souza and Mary Massumi Itoyama.

Cytogenetic evidence for de novo synthesis of rRNA and involvement of nucleolar material in the organization of cell structures during spermiogenesis of Chariesterus armatus (Heteroptera, Coreidae).

The nucleolar material of Chariesterus armatus was analyzed during spermiogenesis in cell preparations impregnated with silver nitrate. Nucleolar corpuscles were observed in spermatids at the beginning of the process, showing that this organoid is also maintained after meiosis. In addition, nucleoli were seen in the round spermatids connected to the X-chromosome (bearer of the nucleolar organizer in C. armatus), indicating de novo synthesis of nucleolar material. This differs from the reorganization of ribosomal granules, transported from meiotic spermatocytes to round spermatids, where they would support protein synthesis, which is reported for other species. We also observed connections of nucleolar corpuscles to the nuclear membrane regions where the tail and the acrosome will be formed, suggesting close involvement of the nucleolar material in the formation of these structures. In addition to the nucleolar bodies, we detected silver-positive structures, which will require new approaches to clarify their role. One of these structures, observed in the cytoplasm, appears to correspond to the chromatoid body, which has been found in several organisms, but is still poorly understood; another is a complex structure to which the tail appears to be connected. We conclude that C. armatus is an appropriate model for understanding not only the synthesis of rRNA in the spermiogenesis, but also the functional meaning of the close relationship of nucleolar material with other structures during this process.

Chagas disease and triatomine biology (Heteroptera, Triatominae), with emphasis on aspects of spermatogenesis

A century after the discovery of Chagas disease, it is still one of the most important parasitic diseases affecting humans. The subfamily Triatominae is important in medical health, because these insects are vectors of Trypanosoma cruzi, the etiologic agent of Chagas disease. These insects are also of important cytological relevance because they have particular cell characteristics, such as persistence of nucleolar material in spermatogenesis. The germ cells of the animal kingdom have chromatoid bodies (CBs) in their cytoplasm that can originate from nucleolar material that is fragmented in the early stages of spermatogenesis and plays an important role in cellular communication between the spermatids during spermiogenesis. Currently, there are few studies on the function and formation of the CB in nucleologenesis, especially with emphasis on the ultrastructure of the cells involved in spermatogenesis of insects. Considering the importance of knowledge about the triatomine fauna, we conducted a study of the biogeography and reports of these insects and a survey of patients with Chagas disease in the northwestern region of São Paulo State. Data collected from 1995 to 2009 indicated 700 individuals with Chagas disease, demonstrating a range of 0 to 40 years, which shows that the disease may be active in this region. Moreover, of the 1150 patients treated for cardiomyopathy, 44% were chagasic. Regarding the triatomines noted and captured in the period from 2004 to 2009, the species were Triatoma sordida and Rhodnius neglectus, with T. sordida being the most abundant. In addition, some triatomines were infected by T. cruzi in various developmental stages. We also analyzed the nucleolar cycle and fibrillarin nucleolar protein expression in CB of spermatogenic cells of T. infestans and T. sordida, using histological, ultrastructural and immunocytochemical techniques. The results revealed fibrillarin nucleolar protein expression in the nucleus and in some cytoplasmic spots of germ cells during spermatogenesis in triatomines. These data suggest that fibrillarin could be a constituent of CB, which was most likely derived from nucleolar fragmentation. This is the first time that fibrillarin protein expression has been shown in CB during spermatogenesis progression in triatomines. Knowledge about the biology of triatomines was deepened in this study and, in particular, the structural and ultrastructural aspects of spermatogenesis in triatomines. This study showed that the disease may be active in the northwestern region of São Paulo and expanded our knowledge of the biology of triatomines, the main vectors of Chagas disease. © FUNPEC-RP.