Trypanosoma Cruzi Isolated From A Triatomine Found In One Of The Biggest Metropolitan Areas Of Latin America

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)To characterize Trypanosoma cruzi (TcI) isolated from a Panstrongylus megistus specimen found in one of the biggest metropolitan areas of Latin America, the relationship between the TcI group of T. cruzi and the transmission cycle in the urban environment was studied. Methods: The T. cruzi strain, Pm, was isolated in a culture medium from the evolutionary forms present in the hindgut of a live male specimen of P. megistus found in the Jabaquara subway in Sao Paulo City. The sample from the triatomine showed trypomastigote forms of Trypanosomatidae, which were inoculated in the peritoneum of Balb/c mice. The sample was then inoculated in Liver Infusion Tryptose medium and J774 cells for the molecular identification and characterization of the parasite. The Pm strain of T. cruzi was identified by isolation in axenic culture medium, and based on the morphology, cell infection, growth kinetics, and molecular characterization. Results: After isolation, the protozoan was identified as T. cruzi. No parasites were detected in the peripheral blood of the animal, which can be a characteristic inherent to the strain of T. cruzi that was isolated. Cell invasion assays were performed in triplicate in the J774 cell line to confirm the invasive ability of the Pm strain and revealed amastigote forms of the parasite within macrophages. Conclusions: Our biological and molecular characterizations helped understand parasite-host interactions and their evolutionary history in context of the associations between vectors, ecotopes, hosts, and groups of the parasite.492183189Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES), Brasilia, DF, BrazilPrograma de Apoio ao Desenvolvimento Cientifico (PADC)Faculdade de Ciencias Farmaceuticas (FCFAR)Campus de Araraquara, Universidade Estadual Paulista (UNESP)Instituto de Biologia (IB), Universidade Estadual de Campinas (UNICAMP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Histological study of spermathecae and trachae of six Triatominae (Hemiptera, Reduviidae)

Orientadores: João Aristeu da Rosa, Estela Sasso CerriDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Os triatomíneos distribuem-se por toda América Latina e apresentam importância parasitológica por serem vetores da doença de Chagas. As várias espécies da subfamília Triatominae (Hemiptera, Reduviidae) possuem similaridades morfológicas, o que dificulta a correta identificação específica com base em caracteres morfológicos externos, principalmente em espécies crípticas. Diante disso este estudo teve como objetivo propor uma alternativa para identificação de seis espécies: Panstrongylus lignarius (Walker, 1837), Panstrongylus megistus (Burmeister, 1835), Rhodnius montenegrensis (Rosa, 2012), Rhodnius prolixus (Stal, 1859), Triatoma infestans (Klug, 1834) e Triatoma tibiamaculata (Pinto, 1926) a partir de estudos morfológicos de traqueias e espermatecas. As traqueias possuem tenídeos na luz traqueal seguido por células epiteliais e externamente à membrana basal, os traqueoblastos estão dispostos aleatoriamente sobre as traqueias. Nos três gêneros as traqueias não mostraram caracteres diferenciais, no entanto foi observado que os traqueoblastos servem como depósito de oxigênio. Panstrongylus megistus e P. lignarius apresentam espermatecas com epitélio simples cilíndrico próximo ao oviduto comum e epitélio simples cúbico alto em sua porção final. A espermateca de Rhodnius montenegrensis é constituída por epitélio pseudoestratificado cilíndrico enquanto que em Rhodnius prolixus apresenta epitélio simples cilíndrico. A espermateca de Triatoma infestans é constituída por epitélio cúbico alto. Triatoma tibiamaculata possui epitélio simples cilíndrico em sua porção inicial e epitélio simples cúbico alto na porção final. Dadas as diferenças anatômicas e histológicas observadas para as seis espécies propõe-se o uso da espermateca para estudos taxionômicos da subfamília TriatominaeAbstract: Triatomines are distributed around all of Latin America and, as vectors of Chagas disease, are of substantial importance in parasitology studies. The many species within the Triatominae subfamily (Hemiptera, Reduviidae) possess morphological similarities, which makes it difficult to identify species correctly based on outer morphological characteristics, particularly in cases of cryptic species. Because of these factors, the objective of this study was to propose an option for distinguishing between six species: Panstrongylus lignarius (Walker 1837), Panstrongylus megistus (Burmeister 1835), Rhodnius montenegrensis (Rosa 2012), Rhodnius prolixus (Stal 1859), Triatoma infestans (Klug 1834), and Triatoma tibiamaculata (Pinto 1926). The method relies on the morphological study of tracheae and spermathecae. The tracheae possess taenidia in the lumen of the trachea surrounded by epithelial cells. Outside the basal membrane, tracheoblasts are randomly available throughout the tracheae. Among the three genera, the trachea did not present differential characteristics; however, it was found that the tracheoblasts serve as a location for oxygen storage. P.megistus and P.lignarius present spermathecae with simple columnar epithelium proximal to the common oviduct and simple tall cubodial epithelium in their end apparatus. The spermatheca of R.montenegrensis consists of pseudostratified columnar epithelium, while in R.prolixus, the spermatheca presents simple columnar epithelium. The spermatheca of T.infestans consists of tall cubodial epithelium. T.tibiamaculata possesses simple columnar epithelium in its initial portion and simple tall cubodial epithelium in its end apparatus. Given the histological and anatomical differences observed among these six species, the use of spermathecae is proposed for taxonomic studies on the Triatominae subfamilyMestradoRelações Antrópicas, Meio Ambiente e ParasitologiaMestra em Biologia AnimalCAPE

Trypanosoma cruzi isolated from a triatomine found in one of the biggest metropolitan areas of Latin America

Abstract: INTRODUCTION: To characterize Trypanosoma cruzi (TcI) isolated from a Panstrongylus megistus specimen found in one of the biggest metropolitan areas of Latin America, the relationship between the TcI group of T. cruzi and the transmission cycle in the urban environment was studied. METHODS: The T. cruzi strain, Pm, was isolated in a culture medium from the evolutionary forms present in the hindgut of a live male specimen of P. megistus found in the Jabaquara subway in São Paulo City. The sample from the triatomine showed trypomastigote forms of Trypanosomatidae, which were inoculated in the peritoneum of Balb/c mice. The sample was then inoculated in Liver Infusion Tryptose medium and J774 cells for the molecular identification and characterization of the parasite. The Pm strain of T. cruzi was identified by isolation in axenic culture medium, and based on the morphology, cell infection, growth kinetics, and molecular characterization. RESULTS: After isolation, the protozoan was identified as T. cruzi. No parasites were detected in the peripheral blood of the animal, which can be a characteristic inherent to the strain of T. cruzi that was isolated. Cell invasion assays were performed in triplicate in the J774 cell line to confirm the invasive ability of the Pm strain and revealed amastigote forms of the parasite within macrophages. CONCLUSIONS: Our biological and molecular characterizations helped understand parasite-host interactions and their evolutionary history in context of the associations between vectors, ecotopes, hosts, and groups of the parasite

Description of the spermathecae and testicles of Triatoma melanocephala Neiva & Pinto, 1923 (Hemiptera: Reduviidae: Triatominae)

Triatoma melanocephala is an endemic species of Brazil. Phenotypic (external morphology) and molecular studies were performed to contribute to the taxonomy and phylogeny of triatomines, since few studies on the internal morphology of these were found. In order to contribute to the biological, morphological and taxonomic knowledge of the subfamily Triatominae, this work describes the internal morphology of the reproductive apparatus of adult males and females of T. melanocephala. For this work 30 specimens from Jequié, Bahia, Brazil were used. Triatoma melanocephala’s reproductive system included slim spermathecae insertions, large/medium rounded terminal bodies, and irregular ellipsoid-shaped testicles with a transparent, vitreous appearance. These results contribute to the knowledge of the reproductive biology of this species of vectorial importance and show taxonomic relevance, as well as possible application in systematic studies

Spermathecae: morphofunctional features and correlation with fat bodies and trachea in six species of vectors of Chagas disease

Since spermatheca is able to transport spermatozoa and maintain a specific microenvironment for the storage of viable sperm cells for long periods of time, specific morphofunctional features must be involved in this capacity, and an efficient nutritional and oxygen supply must be required. In this study, we investigated the histological features of spermathecae and fat bodies in six species of three genera of epidemiological importance for Chagas' disease. The association of the reproductive system with the fat bodies and tracheal system was also focused in these species. The reproductive system, tracheae and fat bodies were fixed in 4% formaldehyde, and embedded in glycol methacrylate. The sections were stained with H.E., picrosirius red and Periodic-Acid Schiff methods for morphological analyses. Paraffin-embedded spermatheca sections were submitted to immunofluorescence for detection of V-ATPase. In P. lignarius, R. montenegrensis and R. prolixus, the spermatheca contains a slightly dilated tubular distal portion. In P. megistus and T. tibiamaculata, the spermatheca shows a large bulbous distal portion, and in T. infestans, a large oval-shaped distal portion. In all species, this portion was surrounded by a thin muscular layer, and the epithelial height varied according to the shape of this terminal portion. All spermathecal proximal portions showed simple columnar epithelium surrounded by a thick muscular layer. The epithelial cells of spermathecae showed PAS-positive cytoplasm and V-ATPase immunofluorescence in the apical surface. Tracheoles and polysaccharide-rich fat body cells were found next or in close contact to the oviduct or spermathecal tissues. The results indicate that the spermatheca proximal portion is related to contraction and sperm transport, whose oxygen and energy supply is guaranteed by the associated tracheal branches and fat bodies. In the storage portion, fat bodies and tracheae seem to be crucial for the maintenance of an optimal spermathecal microenvironment and storage of viable sperm cells. The participation of V-ATPase in the spermathecae epithelial cells may contribute for the maintenance of an optimal luminal milieu to spermatozoa, by alkalinization and/or acidification of lumen, similarly to the other epithelial cell types in insects. Further studies are necessary to clarify the role of this proton pump in the spermathecal epithelial cells197CAPES - Coordenação de Aperfeiçoamento de Pessoal e Nível SuperiorFAPESP – Fundação de Amparo à Pesquisa Do Estado De São Paulosem informação2012/23845-3; 2017/19829-

Characterization of Female External Genitalia and Eggs of Four South American Species of the Triatoma Laporte, 1832 Genus (Hemiptera: Reduviidae: Triatominae)

Triatoma is the most diversified and one of the most important genera from an epidemiological perspective. Given the difficulty in identifying some species of the Triatoma genus, morphological, histological, and morphometric studies were performed to provide new characters that make it possible to differentiate T. garciabesi, T. guasayana, T. patagonica, and T. sordida sensu stricto, triatomines that overlap geographically and have vector potential. Through the external female genitalia, as well as morphology, morphometry, and histology of eggshells, it was possible to discriminate the four species. In addition, this study reinforces the taxonomic validity of T. garciabesi and provides new data for discussion on systematic issues of T. guasayana and T. patagonica

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 analyzed26123131COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPES23038.005285/2011-1

Effects of sodium valproate on the chromatin of triatoma infestans (Klug, 1834) (hemiptera, reduviidae) under in vitro culture conditions

Sodium valproate (VPA) is a classic anticonvulsive, a histone deacetylase inhibitor, and a chromatin remodeling inducer. When injected into specimens of Triatoma infestans, a vector of Chagas disease, VPA affects the chromatin supraorganization of chromocenter heterochromatin in only a few cells of the Malpighian tubules. To test whether this result was explained by the inaccessibility of all of the organ’s cells to the drug, we investigated the nuclear phenotypes and global acetylation of lysine 9 in histone H3 (H3K9ac) in Malpighian tubules cultivated in vitro for 1–24 h in the presence of 0.05 mM–1 mM VPA. The present results revealed that the chromatin decondensation event in the chromocenter body, which was detected only under low VPA concentrations up to a 4-h treatment, was not frequent during organ culture, similar to the results for injected insects. Cultivation of T. infestans Malpighian tubules in vitro for 24 h revealed inadequate for cell preservation even in the absence of the drug. Immunofluorescence signals for H3K9ac following VPA treatment showed a slightly increased intensity in the euchromatin, but were never detected in the chromocenter bodies, except with great intensity at their periphery, where the 18S rDNA is located. In conclusion, when VPA affects the chromocenter heterochromatin in this animal cell model, it occurs through a pathway that excludes a classic global H3K9ac mark. Investigation of nonhistone proteins associated with histone methylation marks is still required to further explain the differential response of T. infestans chromatin to VPA1233CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP421299/2018-5; 307398/2018-8; 304797/2019-7Sem informação2015/10356-2; 2018/07888-

Description of Rhodnius marabaensis sp. n. (Hemiptera, Reduviidae, Triatominae) from Pará state, Brazil

Rhodnius marabaensis sp. n. was collected on 12 May 2014 in the Murumurú Environmental Reserve in the city of Marabá, Pará State, Brazil. This study was based on previous consultation of morphological descriptions of 19 Rhodnius species and compared to the identification key for the genus Rhodnius. The examination included specimens from 18 Rhodnius species held in the Brazilian National and International Triatomine Taxonomy Reference Laboratory in the Oswaldo Cruz Institute in Rio de Janeiro, Brazil. The morphological characteristics of the head, thorax, abdomen, genitalia, and eggs have been determined. Rhodnius prolixus and R. robustus were examined in more detail because the BLAST analysis of a cyt-b sequence shows they are closely related to the new species, which also occurs in the northern region of Brazil. The most notable morphological features that distinguish R. marabaensis sp. n. are the keel-shaped apex of the head, the length of the second segment of the antennae, the shapes of the prosternum, mesosternum and metasternum, the set of spots on the abdomen, the male genitalia, the posterior and ventral surfaces of the external female genitalia, and the morphological characteristics of the eggs. Rhodnius jacundaensis Serra, Serra & Von Atzingen (1980) nomen nudum specimens deposited at the Maraba Cultural Center Foundation - MCCF were examined and considered as a synonym of R. marabaensis sp. n.6214562CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPSem informaçãoSem informação2010/15386-