Climate and Environmental Changes and Their Potential Effects on the Dynamics of Chagas Disease: Hybridization in Rhodniini (Hemiptera, Triatominae).

Chagas disease affects about eight million people. In view of the issues related to the influence of anthropogenic changes in the dynamics of the distribution and reproductive interaction of triatomines, we performed experimental crosses between species of the Rhodniini tribe in order to evaluate interspecific reproductive interactions and hybrid production capacity. Reciprocal crossing experiments were conducted among Rhodnius brethesi × R. pictipes, R. colombiensis × R. ecuadoriensis, R. neivai × R. prolixus, R. robustus × R. prolixus, R. montenegrensis × R. marabaensis; R. montenegrensis × R. robustus, R. prolixus × R. nasutus and R. neglectus × R. milesi. With the exception of crosses between R. pictipes ♀ × R. brethesi ♂, R. ecuadoriensis ♀ × R. colombiensis ♂ and R. prolixus ♀ × R. neivai ♂, all experimental crosses resulted in hybrids. Our results demonstrate that both allopatric and sympatric species produce hybrids, which can generate concern for public health agencies in the face of current anthropogenic events. Thus, we demonstrate that species of the Rhodniini tribe are capable of producing hybrids under laboratory conditions. These results are of great epidemiological importance and raise an important discussion about the influence of climatic and environmental interactions on Chagas disease dynamics.

Revisiting the Hybridization Processes in the Triatoma brasiliensis Complex (Hemiptera, Triatominae): Reproductive Isolation between Triatoma petrocchiae and T. b. brasiliensis and T. lenti.

Triatoma petrocchiae is a species morphologically similar to T. b. brasiliensis (which resulted in a synonymization event); despite this similarity, genetic, morphological, and experimental crossbreeding studies confirmed the specific status of T. petrocchiae. Considering that both species have been reported living in sympatry and that, for a long time, most species of the T. brasiliensis complex were considered only chromatic variants of T. b. brasiliensis, we carried out experimental crosses between T. b. brasiliensis and T. petrocchiae (to confirm whether these species are reproductively isolated) and between T. lenti and T. petrocchiae (to assess whether T. petrocchiae also presents prezygotic isolation with the other species of the T. brasiliensis complex). Reciprocal experimental crosses were conducted, and weekly, the eggs were collected, counted, and separated in new containers to assess the hatch rate. Neither cross resulted in hybrids, demonstrating that there are pre-zygotic reproductive barriers installed between T. petrocchiae and the other species of the T. brasiliensis complex. On the basis of the results above, we demonstrated that T. petrocchiae is reproductively isolated from T. b. brasiliensis and T. lenti. Furthermore, we suggest that T. petrocchiae is the species most derived from the T. brasiliensis complex.

Revisiting the hybridization processes in the Triatoma brasiliensis complex (Hemiptera, Triatominae): Interspecific genomic compatibility point to a possible recent diversification of the species grouped in this monophyletic complex.

Triatomines are hematophagous insects of great epidemiological importance, since they are vectors of the protozoan Trypanosoma cruzi, the etiological agent of Chagas disease. Triatoma brasiliensis complex is a monophyletic group formed by two subspecies and six species: T. b. brasiliensis, T. b. macromelasoma, T. bahiensis, T. juazeirensis, T. lenti, T. melanica, T. petrocchiae and T. sherlocki. The specific status of several species grouped in the T. brasiliensis complex was confirmed from experimental crossing and analysis of reproductive barriers. Thus, we perform interspecific experimental crosses between T. lenti and other species and subspecies of the T. brasiliensis complex and perform morphological analysis of the gonads and cytogenetic analysis in the homeologous chromosomes of the hybrids of first generation (F1). Besides that, we rescue all the literature data associated with the study of reproductive barriers in this monophyletic complex of species and subspecies. For all crosses performed between T. b. brasiliensis, T. b. macromelasoma, T. juazeirensis and T. melanica with T. lenti, interspecific copulas occurred (showing absence of mechanical isolation), hybrids were obtained, none of the male hybrids presented the phenomenon of gonadal dysgenesis and 100% pairing between the chromosomes homeologous of the hybrids was observed. Thus, we demonstrate that there are no pre-zygotic reproductive barriers installed between T. lenti and the species and subspecies of the T. brasiliensis complex. In addition, we demonstrate that the hybrids obtained between these crosses have high genomic compatibility and the absence of gonadal dysgenesis. These results point to reproductive compatibility between T. lenti and species and subspecies of the T. brasiliensis complex (confirming its inclusion in the complex) and lead us to suggest a possible recent diversification of the taxa of this monophyletic group.

Revisiting the Chromosomal Diversification of the Genus Rhodnius (Stål, 1859) (Hemiptera, Triatominae).

Although all triatomines are considered as potential vectors of the Chagas disease, the Triatoma, Panstrongylus, and Rhodnius genera are the most important from the epidemiological point of view. Based on cytogenetic analyzes carried out so far (C banding and FISH), the species of the genus Rhodnius show little interspecific chromosomal variation. Thus, we analyzed the distribution of AT- and CG-rich DNA in the chromatin and chromosomes of the genus Rhodnius and discuss the chromosome evolution of these vectors. Except for Rhodnius domesticus, Rhodnius nasutus, Rhodnius pictipes, Rhodnius colombiensis, and Rhodnius pallescens, all Rhodnius species have euchromatic autosomes with the absence of AT- and CG-rich blocks. Curiously, the same species that have heterochromatin blocks in the autosomes also have chromomycin A3 (CMA3 +) blocks dispersed in the prophasic nucleus (demonstrating that the heterochromatin of these species is rich in CG). Thus, we characterize the AT- and CG-rich DNA pattern for the genus Rhodnius, and we suggest that the pattern of CG-rich heterochromatin in the autosomes of these vectors evolved independently in pallescens, pictipes, and prolixus groups.

Revisiting the genetic variability of Brazilian peridomestic populations of the Chagas disease vector Triatoma sordida () (Hemiptera, Triatominae).

Triatoma sordida is an endemic species to South America, currently considered the species most frequently found in the peridomestic environment in Brazil. This triatomine has a wide ecological tolerability that allows it to inhabit several ecotopes and use different food sources. Although the species is considered predominantly peridomestic, it is also capable of colonizing households and forming numerous intra-household colonies, leading to its inclusion among the priorities for triatomine control campaigns in Brazil. All Brazilian populations of T. sordida are considered as T. sordida sensu stricto by chromosomal analyses (which highlights their epidemiological importance), although molecular studies that characterize the genetic diversity of these populations are scarce, being restricted only to the region of Minas Gerais. Thus, several populations of this vector distributed in the states of Bahia, Goiás, Mato Grosso do Sul, and Minas Gerais were analyzed using mitochondrial markers (cyt b and nd1). Low nucleotide diversity, high haplotypic diversity, low genetic distance, and high FST value were observed, as well as the formation of a monophyletic clade of the Brazilian populations of T. sordida, which confirms that this species has low genetic variability, with all specimens in Brazil grouped in T. sordida sensu stricto. In addition to the genetic and evolutionary importance for the knowledge of the biology of these vectors, these results are important from an epidemiological point of view, thus being able to direct vector control programs.

Reproductive Aspects of Chagas Disease Vectors: Evidence of Transcriptional Activity during the Nucleolar Persistence Phenomenon in the Spermatogenesis of Triatomines.

The subfamily Triatominae currently consists of 154 species, most of them being of great importance for public health because they are considered potential vectors of the protozoan Trypanosoma cruzi, the etiologic agent of Chagas disease. In addition to their epidemiological importance, these insects are considered important biological models for cell studies because they have peculiar characteristics in their cells, for example, persistence of the nucleolus during spermatogenesis. This phenomenon is characterized by the presence of the nucleolus or nucleolar corpuscles during all phases of meiosis. To date, all knowledge is restricted to the study of the presence/absence of the nucleolus during the triatomine meiosis, so the present work aimed to analyze if this persistent nucleolar material has transcriptional activity. Analysis of the meiotic metaphases of Rhodnius montenegrensis and Panstrongylus megistus by using fluorochrome acridine orange made it possible to characterize the presence of RNA in the nucleolar material. Thus, it was demonstrated, for the first time, that the persistent nucleolar material during triatomine meiosis is transcriptionally active, supporting the hypothesis of the relationship between nucleolar persistence during meiosis of these insects and the formation of the chromoid body, an organelle responsible for the support of all transcriptional activities during spermiogenesis.

Cytotaxonomy of Trypanosoma cruzi (Chagas, 1909): Differentiation of T. cruzi I (TcI) and T. cruzi II (TcII) Genotypes Using Cytogenetic Markers.

Chagas disease is a public health problem caused by the Trypanosoma cruzi, and the T. cruzi I (TcI) and T. cruzi II (TcII) groups are considered important genotypes from the clinical point of view. Currently, the groups need to be molecularly analyzed for their identification; thus, we cytogenetically analyzed these groups with the objective of developing more accessible techniques for the characterization of these parasites. TcI and TcII groups were differentiated by nucleus characterization with lacto-acetic orcein (TcI-nucleus with positive heteropycnosis and TcII-nucleus with negative heteropycnosis), emphasizing the importance of the application of this technique for epidemiological and clinical studies of Chagas disease.

CytoKey: Identification Key for the Chagas Disease Vectors of the Largest Brazilian Urban Center (São Paulo State), Based on Cytogenetic Data.

Chagas disease is caused by the protozoan Trypanosoma cruzi. This disease is distributed in 21 Latin American countries, where it is mostly vector-borne. In Brazil, there are 68 triatomine species. To date, the epidemiological surveys indicate that the state of São Paulo presents 11 species of triatomines, and most of these species have already been collected in a home environment and found to be infected with T. cruzi. Problems in correctly identifying species can lead to incorrect panorama of distribution of Chagas disease vectors. Thus, we developed an identification key for the triatomines of the state of São Paulo, based on cytogenetic data. With the exception of Panstrongylus diasi that does not present cytogenetic data available in the literature, all species were differentiated by cytogenetic characteristics. We emphasize the importance of using this key as a simple and objective tool in the entoepidemiological surveys conducted by the vector control programs.

Genetic Structure of Brazilian Populations of Triatoma sordida (Stål, 1859) (Hemiptera, Triatominae) by Means of Chromosomal Markers.

Triatoma sordida is among the main Brazilian species considered as Chagas disease vectors. The genetic studies are directed mainly to phylogenetic questions because this species possibly have suffered cryptic speciation. Furthermore, there are few studies that analyzed the structure and genetic variability of specimens from Brazil and that showed low genetic diversity and strong genetic structuring of the population samples. Therefore, because of great epidemiological importance of T. sordida and mainly the restriction of genetic characterization of this vector only for populations of Minas Gerais state, this article performs a genetic analysis of the T. sordida from seven different Brazilian states (representing different biomes), by means of cytogenetic markers. All analyzed specimens presents the same cytogenetic characteristics: early meiotic prophase with several heterochromatic bodies dispersed in the nucleus (CG-rich), being one of them formed by the associated sex chromosomes surrounded by some autosomal heterochromatic regions, meiotic metaphase with most autosomal pairs exhibiting a C-heterochromatic block in one chromosomal end (CG-rich), Y sex chromosome fully heterochromatin (AT-rich), and X chromosome may present a small C-block (CG-rich). These results are important because the chromosomal markers enable to confirm and expand the low genetic diversity for all Brazilian states occupied by T. sordida, suggesting that all Brazilian populations were originated from a small ancestral population and possibly dispersed to other biomes by founder effect. In addition, we suggest that T. sordida from Brazil are not suffering cryptic speciation and we confirm the classification of all Brazilian examples as T. sordida sensu stricto.

New Evidence of the Monophyletic Relationship of the Genus Psammolestes Bergroth, 1911 (Hemiptera, Reduviidae, Triatominae).

The genus Psammolestes within the subfamily Triatominae and tribe Rhodniini comprises the species Psammolestes arthuri, Psammolestes coreodes, and Psammolestes tertius, all potential vectors of Chagas disease. A feature of Psammolestes is their close association with birds, which makes them an interesting model for evolutionary studies. We analyzed cytogenetically Psammolestes spp., with the aim of contributing to the genetic and evolutionary knowledge of these vectors. All species of the Psammolestes showed the same chromosomal characteristics: chromocenter formed only by sex chromosomes X and Y, karyotype 2n = 22 and constitutive heterochromatin, and AT base pairs restricted to the sex chromosome Y. These results corroborate the monophyly of the genus and lead to the hypothesis that during the derivation of P. tertius, P. coreodes, and P. arthuri from their common ancestor, there was no reorganization in the number or structure of chromosomes.

Parasite-Vector Interaction of Chagas Disease: A Mini-Review.

Trypanosoma cruzi is a protozoan of great importance to public health: it has infected millions of people in the world and is the etiologic agent of Chagas disease, which can cause cardiac and gastrointestinal disorders in patients and may even lead to death. The main vector of transmission of this parasite is triatomine bugs, which have a habit of defecating while feeding on blood and passing the parasite to their own hosts through their feces. Although it has been argued that T. cruzi is not pathogenic for this vector, other studies indicate that the success of the infection depends on several molecules and factors, including the insect's intestinal microbiota, which may experience changes as a result of infection that include decreased fitness. Moreover, the effects of infection depend on the insect species, the parasite strain, and environmental conditions involved. However, the parasite-vector interaction is still underexplored. A deeper understanding of this relationship is an important tool for discovering new approaches to T. cruzi transmission and Chagas disease.

Ultrastructural features of spermatozoa and their phylogenetic application in Zaprionus (Diptera, Drosophilidae).

The genus Zaprionus consists of approximately 60 species of drosophilids that are native to the Afrotropical region. The phylogenetic position of Zaprionus within the Drosophilidae family is still unresolved. In the present study, ultrastructural features of spermatozoa of 6 species of Zaprionus as well as the species Drosophila willistoni and Scaptodrosophila latifasciaeformis were analyzed. The ultrastructure revealed that the species have the same flagellar ultrastructure. Two mitochondrial derivatives, one larger than the other, close to the axoneme were present, primarily in D. willistoni (subgenus Sophophora). Except for Z. davidi and Z. tuberculatus, the analyzed species had paracrystalline material in both mitochondrial derivatives. Moreover, the testes showed 64 spermatozoa per bundle in all of the species. In the cluster analysis, 6 Zaprionus species were grouped closely, but there were some incongruent positions in the cladogram. The results indicated that sperm ultrastructure is an important tool for elucidating the phylogeny and taxonomy of insects.

Immunofluorescence and ultrastructural analysis of the chromatoid body during spermatogenesis of Triatoma platensis and T. rubrovaria (Hemiptera, Triatominae).

This study sought to analyze spermatogenesis in two species of triatomines (Triatoma rubrovaria and T. platensis) by focusing on the chromatoid body (CB) during three stages of spermatogenesis (spermatocytogenesis, meiosis, and spermiogenesis). The cytochemistry technique known as silver impregnation revealed nucleolar persistence. We suggest that this phenomenon is fundamental to the formation of the CB during spermatogenesis, as it allows for the nucleolus or nucleolar fragments to maintain their transcriptional activity during the entire meiosis phase and to apply all transcribed RNA to CB formation. The ultrastructural analysis of T. platensis and T. rubrovaria spermatids revealed the presence of the nucleolus within the spermatid nucleus, as well as the CB near the nuclear membrane. Immunofluorescence for fibrillarin revealed the presence of protein in both the nucleolus and the cytoplasm of spermatogonia. Based on these findings, we suggest that the formation of the CB begins during the first phase of spermatogenesis, or spermatocytogenesis. Furthermore, we also observed the presence of fibrillarin protein in the CB near the elongating spermatids. Unlike the spermatogonia, spermatids showed no fibrillarin markings in the nucleolar region, a finding which is consistent with the lack of post-meiotic transcriptional activity during triatomine spermiogenesis. Thus, this study suggests that the formation of the CB begins during spermatocytogenesis and is intensified by transcriptional activity when nucleolar persistence occurs in meiosis. Moreover, the findings are consistent with the absence of transcriptional activity in the nucleolus during spermiogenesis, and they demonstrate that all transcriptional activity during spermatid differentiation is supported by the CB.

Análise das possíveis regiões organizadoras nucleolares e da atividade nucleolar em Triatoma melanocephala e T. lenti, importantes vetores da doença de Chagas / Analysis of nucleolus organizer regions and nucleolar activity in important vectors of Chagas disease (Triatoma melanocephala and T. lenti)

Triatoma melanocephalae T. lentisão espécies crípticas de triatomíneos pertencentes ao subcomplexo Brasiliensis. Esses organismos foram agrupados no subcomplexo apenas por caracteres morfológicos e pela disposição geográfica. Sendo assim, estudos citogenéticos são considerados como importantes ferramentas na classificação dos triatomíneos e, com isso, podem auxiliar na criação de um plano de profilaxia da doença. Por meio da técnica citogenética de impregnação por íons prata, foi possível visualizar a atividade nucleolar e as Regiões Organizadoras Nucleolares (RONs) desses insetos. T. melanocephala apresentou três RONs ativas nos autossomos durante a prófase I. T. lenti apresentou duas RONs ativas nos autossomos durante a prófase I e a metáfase I. Ambas as espécies apresentaram o fenômeno de persistência do material nucleolar encontrado em triatomíneos. Sendo assim, por meio da análise das RONs, foi possível observar que T. lenti, quando comparado com os outros organismos do subcomplexo, apresentou marcações semelhantes à T. tibiamaculata e que T. melanocephala não apresenta nenhuma relação direta com o subcomplexo.

Triatoma melanocephala and T. lenti are important vectors of Chagas disease. These cryptic species of triatomines are grouped in the subcomplex brasiliensis due only to morphological characters and geographical distribution. Cytogenetic studies are important to the classification of insects and can assist in creating a disease prevention plan. The aim of the present study was to determine nucleolar activity and nucleolus organizer regions (NORs) in these insects using the cytogenetic method of silver ion impregnation. T. melanocephala exhibited three active NORs in autosomes during prophase I. T. lentiexhibited two active NORs in autosomes during prophase I and metaphase I. Both species exhibit the persistent nucleolar material found in triatomines. The analysis of NORs in the present study revealed that T. lentiexhibited labeling similar to that found in T. tibiamaculata, which belongs to the subcomplex, whereas T. melanocephala shows no direct relationship with the subcomplex.

Nucleolar cycle and chromatoid body formation: is there a relationship between these two processes during spermatogenesis of Dendropsophus minutus (Amphibia, Anura)?

The goals of this study were to monitor the nucleolar material distribution during Dendropsophus minutus spermatogenesis using cytological and cytochemical techniques and ultrastructural analysis, as well as to compare the nucleolar material distribution to the formation of the chromatoid body (CB) in the germ epithelium of this amphibian species. Nucleolar fragmentation occurred during the pachytene of prophase I and nucleolus reorganization occurred in the early spermatid nucleus. The area of the spermatogonia nucleolus was significantly larger than that of the earlier spermatid nucleolus. Ultrastructural analysis showed an accumulation of nuages in the spermatogonia cytoplasm, which form the CB before nucleolar fragmentation. The CB was observed in association with mitochondrial clusters in the cytoplasm of primary spermatocytes, as well as in those of earlier spermatids. In conclusion, the nucleolus seems to be related to CB formation during spermatogenesis of D. minutus, because, at the moment of nucleolus fragmentation in the primary spermatocytes, the CB area reaches a considerable size and is able to execute its important functions during spermatogenesis. The reorganized nucleolus of the earlier spermatids has a smaller area due to several factors, among them the probable migration of nucleolar fragments from the nucleus to the cytoplasm, and plays a part in the CB chemical composition.

Spermatogenesis and nucleolar activity in Triatoma klugi (triatomine, Heteroptera)

Triatoma klugi is a Chagas disease vector in the Rio Grande do Sul State. Triatominae chromosomes are holocentric and sex chromosomes segregation is post-reductional. In this paper we describe the karyotype of male T. klugi and a meiotic analysis including the nucleolar behavior during spermatogenesis. Testis cells were analyzed after lacto-acetic orcein and silver nitrate staining. Two autosomes and the heterochromosomes presented nucleolar activity (Ag-NORs) during diplotene-diakinesis. The analysis of metaphase I and II revealed a karyotype with 2n = 20+XY. In metaphase I a prominent nucleolar mass was observed in the cell periphery and small silver grains were detected in metaphase II. During anaphase, the chromosomes segregated in parallel and a typical holocentric late migration behavior was observed. The restoration of the nucleolus was an important feature in this phase. During telophase nucleolar masses persisted and in early spermiogenesis the spermatids presented a small peripheral mass until elongation. The present study is a contribution to the study of chromatin behavior and nucleolar persistence in meiosis.

Estrutura nucleolar e impregnaçäo por íons prata / Nucleolar structure and impregnation by silver ions

Os principais aspectos estruturais,fisiológicos e citoquímicos,ligados ao nucléolo,importante organela nuclear,responsável pela biogênese ribossômica e,consequentemente,pela síntese proteíca celular,são revisados no presente artigo. A identificação dessa organela é feita,classicamente,através da técnica de impregnação por íons prata,tanto em materiais biológicos preparados para observação em microscopia óptica,como para eletrônica. Apesar da grande quantidade de dados contidos na literatura,baseados no uso da impregnação pela prata,até recentemente,a verdadeira natureza citoquímica desse processo de nucleolares,B23(numatrina) e C23(nucleolina), e à associação dessa última ao DNA ribossômico(DNAr),a especificidade dessa coloração