Cytotaxonomy of Dipetalogaster maxima Uhler, 1894 (Hemiptera, Reduviidae, Triatominae) / Citotaxonomia de Dipetalogaster maxima Uhler, 1894 (Hemiptera, Reduviidae, Triatominae)

Abstract The Triatomini tribe consists of ten genera and is regarded as one of the most important tribes from epidemiological point of view. The genus Dipetalogaster Usinger, 1939 is composed only by the species Dipetalogaster maxima Uhler, 1894. This triatomine is exclusive of the Mexico and is a potential vector for Chagas disease. Besides the epidemiological importance, the insects of the Triatominae subfamily are important biological models for cytogenetic studies. Therefore, in order to contribute to the knowledge on the reproductive biology and assist in citotaxonomy of D. maxima, this study aimed to describe spermatogenesis, as well as confirm the karyotype and heterochromatic patterns of this Mexican triatomine species. The seminiferous tubules were torn, fixed to a cover slip and underwent the cytogenetic technique of Lacto-acetic orcein and C-banding. Through the cytogenetics analysis of testicular material D. maxima it was possible to confirm the karyotype (2n = 22), describe the stages of spermatogenesis and characterize the heterochromatic pattern (restricted to sex chromosome Y) of the species. D. maxima showed the same arrangement of heterochromatin described for Triatoma lecticularia (Stål, 1859) (a species that occur in United States of American and Mexico and is phylogenetically related with D. maxima), highlighting the importance of this analysis as an optimization tool to explore phylogenetic correlations.

Resumo A tribo Triatomini consiste em dez gêneros e é considerada uma das tribos mais importantes do ponto de vista epidemiológico. O gênero Dipetalogaster Usinger, 1939 é composto apenas pela espécie Dipetalogaster maxima Uhler, 1894. Este triatomíneo é exclusivo do México e é um vetor potencial da doença de Chagas. Além da importância epidemiológica, os insetos da subfamília Triatominae são importantes modelos biológicos para estudos citogenéticos. Portanto, a fim de contribuir para o conhecimento da biologia reprodutiva e complementar o conceito específico de D. maxima, este trabalho objetivou descrever a espermatogênese, bem como confirmar o padrão cariotípico e heterocromático desta espécie mexicana, com foco citotaxonômico. Os túbulos seminíferos foram dilacerados, fixados em uma lamínula e submetidos à técnica citogenética de Orceína lacto-acética e Bandamento-C. Por meio da análise citogenética do material testicular de D. maxima foi possível confirmar o cariótipo (2n = 22), descrever os estágios da espermatogênese e caracterizar o padrão heterocromático (restrito ao cromossomo Y sexual) da espécie. D. maxima apresentou o mesmo arranjo de heterocromatina descrito para Triatoma lecticularia (Stål, 1859) (espécie que ocorre no México e nos Estados Unidos da América, filogeneticamente relacionada com D. maxima), destacando a importância desta técnica como ferramenta para explorar correlações filogenéticas.

Cytotaxonomy of Dipetalogaster maxima Uhler, 1894 (Hemiptera, Reduviidae, Triatominae)

Abstract The Triatomini tribe consists of ten genera and is regarded as one of the most important tribes from epidemiological point of view. The genus Dipetalogaster Usinger, 1939 is composed only by the species Dipetalogaster maxima Uhler, 1894. This triatomine is exclusive of the Mexico and is a potential vector for Chagas disease. Besides the epidemiological importance, the insects of the Triatominae subfamily are important biological models for cytogenetic studies. Therefore, in order to contribute to the knowledge on the reproductive biology and assist in citotaxonomy of D. maxima, this study aimed to describe spermatogenesis, as well as confirm the karyotype and heterochromatic patterns of this Mexican triatomine species. The seminiferous tubules were torn, fixed to a cover slip and underwent the cytogenetic technique of Lacto-acetic orcein and C-banding. Through the cytogenetics analysis of testicular material D. maxima it was possible to confirm the karyotype (2n = 22), describe the stages of spermatogenesis and characterize the heterochromatic pattern (restricted to sex chromosome Y) of the species. D. maxima showed the same arrangement of heterochromatin described for Triatoma lecticularia (Stål, 1859) (a species that occur in United States of American and Mexico and is phylogenetically related with D. maxima), highlighting the importance of this analysis as an optimization tool to explore phylogenetic correlations.

Resumo A tribo Triatomini consiste em dez gêneros e é considerada uma das tribos mais importantes do ponto de vista epidemiológico. O gênero Dipetalogaster Usinger, 1939 é composto apenas pela espécie Dipetalogaster maxima Uhler, 1894. Este triatomíneo é exclusivo do México e é um vetor potencial da doença de Chagas. Além da importância epidemiológica, os insetos da subfamília Triatominae são importantes modelos biológicos para estudos citogenéticos. Portanto, a fim de contribuir para o conhecimento da biologia reprodutiva e complementar o conceito específico de D. maxima, este trabalho objetivou descrever a espermatogênese, bem como confirmar o padrão cariotípico e heterocromático desta espécie mexicana, com foco citotaxonômico. Os túbulos seminíferos foram dilacerados, fixados em uma lamínula e submetidos à técnica citogenética de Orceína lacto-acética e Bandamento-C. Por meio da análise citogenética do material testicular de D. maxima foi possível confirmar o cariótipo (2n = 22), descrever os estágios da espermatogênese e caracterizar o padrão heterocromático (restrito ao cromossomo Y sexual) da espécie. D. maxima apresentou o mesmo arranjo de heterocromatina descrito para Triatoma lecticularia (Stål, 1859) (espécie que ocorre no México e nos Estados Unidos da América, filogeneticamente relacionada com D. maxima), destacando a importância desta técnica como ferramenta para explorar correlações filogenéticas.

First cytogenetic study of Cavernicola pilosa Barber, 1937 (Hemiptera, Triatominae).

Cavernicola pilosa is a triatomine species that lives in caves and feeds on bat blood. This vector has a wide geographical distribution, and is found in Brazil, Colombia, Panama, Peru, and Venezuela. Little is known about the reproductive biology of this species, because most previous studies have only characterized its morphology, morphometry, ecology, and epidemiology. Therefore, this study aimed to obtain preliminary data related to spermatogenesis in C. pilosa by conducting cytogenetic analysis. Analysis of the heterochromatic pattern of C. pilosa during the initial prophases revealed that heterochromatic blocks are only present in the sex chromosomes. Based on the analyses of the meiotic metaphase and prophases, we found that the sex determination system of C. pilosa is XY and the chromosomes are holocentric. C. pilosa spermatids are filamentous and have long flagella. It was not possible to detect corpuscle or filament heteropycnosis in spermatids of this species. The initial cytogenetic data presented in this study are important in characterizing the spermatogenesis and heterochromatic patterns of C. pilosa. Our results suggest that adaptation to troglodytism did not result in differences in spermatogenesis in this vector.

Spermatogenesis and nucleolar behavior in Triatoma vandae and Triatoma williami (Hemiptera, Triatominae).

This study describes spermatogenesis in Triatoma vandae and the nucleolar behavior of T. vandae and Triatoma williami, with a cytotaxonomic focus. Analysis of mitotic and meiotic metaphases of T.vandae confirms the species karyotype. T. vandae presents some characteristics during meiosis that differentiate it from T. williami, including the presence of a chromocenter with two sex chromosomes individualized during early prophase, and the presence of a bi- or tripartite corpuscle inpolyploid nuclei. It was possible to observe the compaction of chromatin during prophase resulting in holocentric chromosomes. During metaphase,the autosomes presented a ring shape and the sex chromosomes were in the center of the ring. These chromosomes were separated in anaphase. Although it is common, we did not observe the phenomenon of late migration of the sex chromosomes. By means of silver ion impregnation it was possible to describe nucleologenesis in T. vandae and T. williami. In both species we observed persistence of the nucleolar material duringmeiosis. In addition to the cells in meiotic division, we also observed the presence of polyploid nuclei in the seminiferous tubule walls that nourish the cells during cell division. The nucleolar markings reflect their capacity for synthetic activity. T. vandae and T. williami presented only one nucleolar corpuscle, which reflects low synthetic activity. This study confirms the karyotype of T. vandae, describes characteristics that differentiate T. vandae and T. williami during meiosis, and describes the phenomenon of nucleolar persistence in both species.

Nucleolar activity during larval development of Myrmeleon uniformis Navas, 1920 (Neuroptera, Myrmeleontidae).

It has been reported in the literature that the Malpighian tubules of Neuroptera in the third instar undergo drastic histological changes, when they stop functioning in osmoregulation and start to secrete silk fibers for a cocoon. Therefore, to increase our knowledge about these cellular alterations that occur in the larvae of Neuroptera, we analyzed the cells that constitute the Malpighian tubules of each larval instar of the species Myrmeleon uniformis, with emphasis on nucleolar activity. Malpighian tubules, after being removed, were fixed on a slide using liquid nitrogen and stained by silver impregnation. In addition, total protein of the tubules was quantified. By analyzing the cells in the first instar larval stage, we observed only two silver-stained nucleolar regions. In cells of second instar larvae, there was an increase in the number of stained regions, and in the third instar, the number of nucleolar regions was very large. Agarose gel electrophoresis indicated that third instar larvae had high synthetic activity, where the total amount of proteins was larger in third instar stage than in the other larval stages. Furthermore, the most abundant proteins displayed molecular weights of about 32-43 kDa and were probably precursors of silk fibers. Thus, the results obtained showed that nucleolar alterations occur in the cells of the Malpighian tubules of larval instars of M. uniformis and this is directly related to the production of silk fibers used by the pupa to ensure the completion of metamorphosis.

Spermatogenesis in Triatoma melanocephala (Hemiptera: Triatominae).

Triatoma melanocephala is a rare species of Hemiptera. It belonged to the Brasiliensis subcomplex and presents morphological characteristics very close to those of Triatoma vitticeps. We investigated spermatogenesis of T. melanocephala and compared it with that of T. vitticeps in order to determine whether these organisms have similar cytogenetic characteristics. Lacto-acetic orcein staining was used to observe all stages of meiosis. These two species were found to have the same karyotype (2n = 20A + X1X2X3Y), heteropycnotic corpuscles in the polyploid spermatogonial cells, interfasic and profasic nucleus, and chromocenter with four sex chromosomes during prophase. Thus, we conclude that besides the morphologic similarity of T. melanocephala with T. vitticeps, they also have similar spermatogenesis and cytogenetics.

Microdissection testicular sperm extraction causes spermatogenic alterations in the contralateral testis.

Testicular sperm extraction (TESE) associated with intracytoplasmic sperm injection has allowed many men presenting non-obstructive azoospermia to achieve fatherhood. Microdissection TESE (microTESE) was proposed as a method to improve sperm retrieval rates in these patients; however, there have been failures. Little is known about whether microTESE leads to spermatogenic alterations in the contralateral testis. We assessed histological outcomes of experimental microTESE in the contralateral testis of adult male rabbits. Nine adult male rabbits were divided into three groups: control (testicular biopsy to observe normal histological and morphometric values), sham (incision of the tunica vaginalis, and a contralateral testicular biopsy to observe histological and morphometric patterns, 45 days later), and study (left testicular microTESE, and a right testicular biopsy to observe histological and morphometric patterns, 45 days later). Sections were assessed by calculating Johnsen-like scores, and measuring total tubule diameter, lumen diameter and epithelial height. The results were compared using ANOVA and Bonferroni's statistical analysis. Morphometric evaluation of the seminiferous tubules did not demonstrate differences between the three groups. However, microTESE caused spermatogenic alterations, leading to maturation arrest in the contralateral testis.

Cytochemical characterization of Triatoma infestans and Panstrongylus megistus salivary gland cells (Hemiptera, Reduviidae, Triatominae).

The Triatominae subfamily has medical sanitary importance, since these insects are vectors of Trypanosoma cruzi, etiologic agent of Chagas Disease, and Trypanosoma rangeli, which develops in the salivary glands and it is frequently found in mixed infections with T. cruzi. Triatomines of Triatoma and Panstrongylus genera possess a salivary gland complex composed of a pair with three well differentiated units: the anterior (D1), median (D2) and posterior (D3). Saliva is secreted during blood meal and antagonizes hemostatic, inflammatory and immunological systems imposed by the host, which facilitate the hematophagy. In order to identify nuclear structures, we studied interphase nuclei of salivary gland cells of adult insects, males and females, of Triatoma infestans and Panstrongylus megistus. The glands were removed from insects, fixed in acetic acid (45%) and lactic acid (50%), squashed 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 preparations were examined with a Zeiss Jenaval photomicroscope and photographed. The results evidenced predominance of binucleated cells in D1 and D2 glands and mononucleated ones in D3. In all salivary glands were observed bulky and polyploid nucleus, a clear association between nucleolar and heterochromatic corpuscles, cytoplasmatic metachromasy and many pre-secretion vesicles in cytoplasm. Such characteristics were associated with intense synthesis activity to produce the saliva. Species were mainly differentiated by a larger heterochromatic corpuscle observed only in T. infestans (called as chromocenter), while P. megistus showed a predominance of euchromatin, with some heterochromatic corpuscles just in males. Females of both species showed a smaller quantity of heterochromatin, which could be related to the high metabolism because of the oviposition.

Programmed cell death in salivary glands of Drosophila arizonae and Drosophila mulleri

Programmed cell death (PCD) in insect metamorphosis assumes a great diversity of morphology and controlling processes that are still not well understood. With the objective of obtaining information about the PCD process, salivary glands of Drosophila arizonae and D. mulleri were studied during larval-pupal development. From the results, it can be concluded that the type of the PCD that occurs in these organs is morphologically typical of apoptosis (formation of apoptotic nuclei, followed by fragmentation into apoptotic bodies). Histolysis happens in both species, between 22 and 23 h after pupation. There were no significant differences between the species studied. Apoptosis does not occur simultaneously in all cells. Cytoplasmic acid phosphatase activity gradually increases during development, suggesting the existence of acid phosphatases that are only expressed during the apoptotic stage. Twenty hours after pupation, salivary glands already show biochemical alterations relative to nuclear permeability such as acidification, possibly due to the fusion of lysosomes with the nucleus a few hours before apoptosis. Autophagy seems to act together with apoptosis and has a secondary role in cell death.

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.

Acid phosphatase activity distribution in salivary glands of triatomines (Heteroptera, Reduviidae, Triatominae).

Acid phosphatase activity (Gömori technique) in salivary gland cells was investigated in adult insects (males and females) of four species of triatomines: Triatoma infestans, Panstrongylus megistus, Rhodnius neglectus, and Rhodnius prolixus. Binucleated cells with bulky and polyploidy nuclei were detected, with acid phosphatase activity in the heterochromatin and nucleolus, which showed the most intense response. Thus, the activity of these phosphatases during rRNA molecule transcription, possibly in the nucleolar fibrillar center, is suggested. The difference in reactivity found among salivary glands is associated with the cellular metabolism of these regions and, probably, with the biosynthesis of their different secretions. This must be essential in maintaining the hematophagy of triatomines.

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 cma3/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, CMA3 staining, and FISH, confirming that these chromosomes bear nucleolar organizer regions (NORs). In P. megistus, two of the three sex chromosomes were CMA3/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.

Phylogenetic approach to the study of Triatomines (Triatominae, Heteroptera).

Triatomines are insects belonging to the Hemiptera order, Heteroptera suborder, Reduviidae family and Triatominae subfamily. All members of this subfamily are hematophagous. Triatomines evolved from Reduviidae predators and they are probably polyphyletic in origin. The combination of anatomical, physiological and ethological factors observed in this group, as well as the plesiomorphic and apomorphic characters that differentiate the five tribes and fourteen triatomine genera reinforce the polyphiletic hypotesis. However if we consider the five groups of triatomines, the Rhodniini, Cavernicolini, Bolboderini, Linshcosteini and Alberproseniini tribes constitute monophyletic groups, while the Triatomini tribe is considered polyphyletic. The New World is the center of triatomine diversity and seems to be the point of group origin. Of approximately 137 triatomine species, 105 are only found in the Americas. It is now considered that triatomines represent a polyphyletic group defined according to their convergent apomorphic hematophagous characters, which have appeared several times in Reduviidae. This study revises the phylogeny of these vectors of Chagas' disease, covering such topics as the origin of hematophagy in triatomines and ancestral proposal for the group.

Phylogenetic approach to the study of Triatomines (Triatominae, Heteroptera)

Os triatomíneos são insetos pertencentes à ordem Hemiptera, subordem Heteroptera, família Reduviidae e subfamília Triatominae. Todos os membros desta subfamília são hematófagos. Os triatomíneos surgiram a partir de reduvídeos predadores e provavelmente têm origem polifilética. A combinação dos fatores anatômicos, fisiológicos e etológicos presentes no grupo, bem como os caracteres plésio e apomórficos que diferenciam as cinco tribos e os quatorze gêneros de triatomíneos reforçam a hipótese polifilética. As tribos Rhodniini, Cavernicolini, Bolboderini, Linshcosteini e Alberproseniini constituem grupos monofiléticos, per si, enquanto a tribo Triatomini é considerada polifilética. O Novo Mundo é claramente o centro de diversidade dos triatomíneos e possivelmente é a região de sua origem. Entre as aproximadamente 129 espécies desses insetos, 105 ocorrem somente nas Américas. Atualmente, os triatomíneos são considerados um grupo polifilético, definido com base em seus caracteres apomórficos convergentes relacionados à hematofagia. Acredita-se que este hábito alimentar tenha surgido várias vezes nos Reduviidae durante sua evolução. O presente trabalho faz uma revisão sobre a filogenia destes vetores da Doença de Chagas, aborda tópicos como a origem da hematofagia nos triatomíneos e ancestralidade proposta para o grupo.