Genes encoding defensins of important Chagas disease vectors used for phylogenetic studies.

Insects possess both cellular and humoral immune responses. The latter makes them capable to recognize and control invading pathogens after synthesis of a variety of small proteins, also known as antimicrobial peptides. Defensins, cysteine-rich cationic peptides with major activity against Gram-positive bacteria, are one ubiquitous class of antimicrobial peptides, widely distributed in different animal and plant taxa. Regarding triatomines in each of the so far analyzed species, various defensin gene isoforms have been identified. In the present study, these genes were sequenced and used as a molecular marker for phylogenetic analysis. Considering the vectors of Chagas disease the authors are reporting for the first time the presence of these genes in Triatoma sordida (Stål, 1859), Rhodnius nasutus (Stål, 1859), and Panstrongylus megistus (Burmeister, 1835). Members of the Triatoma brasiliensis species complex were included into the study to verify the genetic variability within these taxa. Mainly in their mature peptide, the deduced defensin amino acid sequences were highly conserved. In the dendrogram based on defensin encoding nucleotide, sequences the Triatoma Def3/4 genes were separated from the rest. In the dendrogram based on deduced amino acid sequences the Triatoma Def2/3/4 together with Rhodnius DefA/B pre-propeptides were separated from the rest. In the sub-branches of both the DNA and amino acid dendrograms, the genus Triatoma was separated from the genus Rhodnius as well as from P. megistus.

Comparative descriptions of eggs from three species of Rhodnius (Hemiptera: Reduviidae: Triatominae).

The authors describe and compare the morphological and ultrastructural characteristics of eggs from the three most recent described species of the genus Rhodnius Stål, 1859, which have not previously been studied. These species are Rhodnius colombiensis (Mejia, Galvão & Jurberg 1999), Rhodnius milesi (Carcavallo, Rocha, Galvão & Jurberg 2001) and Rhodnius stali (Lent, Jurberg & Galvão 1993). The results revealed that there are similarities in the exochorial architecture of optical microscopy and scanning electron microscopy; these include the predominance of hexagonal cells that are common to all Rhodnius species and variable degrees of lateral flattening, which is common not only to species of this genus, but also to the Rhodniini tribe. Differences in overall colour, the presence of a collar in R. milesi, a longitudinal bevel in R. stali and the precise length of R. colombiensis can be useful distinguishing features. As a result of this study, the key for egg identification proposed by Barata in 1981 can be updated.

Triatoma infestans relies on salivary lysophosphatidylcholine to enhance Trypanosoma cruzi transmission.

Triatoma infestans is a mandatory haematophagous vector of Chagas disease in Brazil. Despite a large number of studies on the anti-haemostatic molecules present in its saliva, the role of its salivary components on parasite transmission is poorly understood. Here, we show that the bioactive lipid molecule, lysophosphatidylcholine (LPC), is present in the salivary gland of T. infestans. We characterized the lipid profiles of each unit of the T. infestans salivary gland. We noticed that LPC is present in the three units of the salivary gland and that the insect feeding state does not influence its proportion. T. infestans saliva and LPC can enhance T. cruzi transmission to mice by dramatically altering the profile of inflammatory cells at the site of inoculation on mouse skin, facilitating the transmission of T. cruzi to the vertebrate host. Consequently, the mortality curves of either saliva- or LPC-injected mice display significant higher mortality rates than the control. Altogether, these results implicate LPC as one of key salivary molecule involved in Chagas disease transmission.

Bacterial community composition in the salivary glands of triatomines (Hemiptera: Reduviidae).

BACKGROUND: Chagas disease is caused by the parasite Trypanosoma cruzi and is transmitted through triatomines (Hemiptera: Reduviidae). In the last year, many studies of triatomine gut microbiota have outlined its potential role in modulating vector competence. However, little is known about the microbiota present in the salivary glands of triatomines. Bacterial composition of salivary glands in selected triatomine species was investigated, as well as environmental influences on the acquisition of bacterial communities. METHODOLOGY/PRINCIPAL FINDINGS: The diversity of the bacterial communities of 30 pairs of salivary glands of triatomines was studied by sequencing of the V1- V3 variable region of the 16S rRNA using the MiSeq platform (Illumina), and bacteria isolated from skin of three vertebrate hosts were identified based on 16S rRNA gene sequence analysis (targeting the V3-V5 region). In a comparative analysis of microbiota in the salivary glands of triatomine species, operational taxonomic units belonging to Arsenophonous appeared as dominant in Triatoma spp (74% of the total 16S coverage), while these units belonging to unclassified Enterobacteriaceae were dominant in the Rhodnius spp (57% of the total 16S coverage). Some intraspecific changes in the composition of the triatomine microbiota were observed, suggesting that some bacteria may have been acquired from the environment. CONCLUSIONS AND SIGNIFICANCE: Our study revealed the presence of a low-diversity microbiota associated to the salivary glands of the evaluated triatomines. The predominant bacteria genera are associated with triatomine genera and the bacteria can be acquired in the environment in which the insects reside. Further studies are necessary to determine the influence of bacterial communities on vector competence.

[Intra-domiciliary capture of Panstrongylus rufotuberculatus (Champion, 1899) (Hemiptera, Reduviidae, Triatominae) in Piura, Peru]. / Hallazgo de Panstrongylus rufotuberculatus (Champion, 1899) (Hemiptera, Reduviidae, Triatominae) en ambiente domiciliario en la Región Piura, Perú.

Panstrongylus rufotuberculatus is widely distributed in Central and South America. In Peru, it has been found in Tumbes, Piura, and Cuzco. The authors report the presence of P. rufotuberculatus in Chirinos-La Pareja, Suyo district, Ayabaca Province, Piura Region, Peru. Twenty-eight specimens were collected in two of 15 dwellings: three 2nd instar, four 3rd instar, two 4th instar, and eight 5th instar nymphs, three male and eight female, in an intra-domiciliary colony of guinea pigs (Cavia porcellus, raised indoors as a source of diet protein) and beds and bedroom walls. None of the fecal samples were naturally infected with Trypanosoma cruzi. This is the first report of synanthropic presence of P. rufotuberculatus in the region. The finding emphasizes the need for careful entomological and epidemiological surveillance of this and other triatomine species in the Region.

Description of the feeding preferences of triatominae in the Chagas disease surveillance study for the State of Pernambuco, Brazil (Hemiptera: Reduviidae).

INTRODUCTION:: Studying the feeding preferences of triatomines is an important entomological surveillance tool, since continuous surveillance of the disease is necessary. METHODS:: The precipitin reaction was used to describe the feeding preferences of triatomines along with their natural infection by flagellates similar to Tyrpanosoma cruzi. Six hundred eighty-seven insects were examined, including Triatoma brasiliensis, Triatoma pseudomaculata, and Panstrongylus lutzi. RESULTS:: Sixty-nine (10%) of 687 triatomines examined tested positive for flagellates similar to T. cruzi, and 8 (1.2%) of these fed on human blood. CONCLUSIONS:: This study found potential transmitters of Chagas disease both inside and outside the domiciliar environment.

Mitochondrial Gene Confirms the Specific Status of Triatoma pintodiasi Jurberg, Cunha, and Rocha, 2013 (Hemiptera, Triatominae), an Endemic Species in Brazil.

Chagas disease is most frequently transmitted to humans through contact with feces of insects from the Triatominae subfamily. In Brazil, there are 65 species of triatomines distributed throughout the country's 27 states. Among the species in the state of Rio Grande do Sul, Triatoma rubrovaria, Triatoma oliveirai, Triatoma pintodiasi, Triatoma klugi, Triatoma carcavalloi, and Triatoma circummaculata (with the addition Triatoma limai, which is endemic to Argentina) form the T. rubrovaria subcomplex. The last species described and grouped into this subcomplex was T. pintodiasi Thus, this study characterized the genetic distance between T. pintodiasi and of the other members of the T. rubrovaria subcomplex to evaluate the specific status of T. pintodiasi The genetic distance observed between T. pintodiasi and the other species of the T. rubrovaria subcomplex was large, a finding which highlights the specific status of the species considered to be cryptic of T. circummaculata.

[Feeding sources evaluation of Panstrongylus lutzi (Neiva & Pinto, 1923) (Hemiptera: Reduviidae: Triatominae) in the State of Ceará]. / Estudo das fontes alimentares de Panstrongylus lutzi (Neiva & Pinto, 1923) (Hemiptera: Reduviidae: Triatominae) no Estado do Ceará.

The authors used precipitin technique to detect the feeding sources of Panstrongylus lutzi (Neiva & Pinto, 1923) in 20 municipalities of Ceará State, Brazil. The results detected the presence of blood from eight different blood sources and mixed feedings, demonstrating that Panstrongylus lutzi is an eclectic species. High infection rates for Trypanosoma like-cruzi were detected.

Triatominae (Hemiptera, Reduviidae) in the Pantanal region: association with Trypanosoma cruzi, different habitats and vertebrate hosts.

INTRODUCTION: The transmission cycle of Trypanosoma cruzi in the Brazilian Pantanal region has been studied during the last decade. Although considerable knowledge is available regarding the mammalian hosts infected by T. cruzi in this wetland, no studies have investigated its vectors in this region. This study aimed to investigate the presence of sylvatic triatomine species in different habitats of the Brazilian Pantanal region and to correlate their presence with the occurrences of vertebrate hosts and T. cruzi infection. METHODS: The fieldwork involved passive search by using light traps and Noireau traps and active search by visual inspection. The light traps were placed at five selected points along forested areas for seven nights during each of the nine excursions. At each point where a light trap was set, eight Noireau traps were placed in palm trees and bromeliads. RESULTS: In all, 88 triatomine bugs were collected: two and one individuals from light traps and Noireau traps, respectively; three from peridomestic areas; 23 in coati nests; and 59 in thornbird nests. In this study, active search in microhabitats showed higher efficiency than passive search, since 95% of the triatomine bugs were caught in nests. Further, triatomine bugs were only found to be infected by T. cruzi in coati nests. CONCLUSIONS: Coati nests might act as a point of convergence and dispersion for triatomine bugs and mammal hosts infected by T. cruzi, thereby playing an important role in the sylvatic cycle of T. cruziin the Pantanal region.

Karyosystematics of Triatoma rubrofasciata (De Geer, 1773) (Hemiptera: Reduviidae: Triatominae).

Triatoma rubrofasciata (De Geer) is the first species of triatomine described, and little is known on its vector biology. Studies are restricted to starvation resistance, interspecific morphometric variability, morphometry of testis follicles, coloration of the testicular peritoneal sheath, ultrastructure of the male accessory glands, phylogeny and cytogenetics. Thus, this study aims to address the karyosystematics of T. rubrofasciata and the possible events related to karyotype evolution of this species. Four adult males were analyzed cytogenetically. The analysis of meiotic metaphases of T. rubrofasciata allowed to confirm the karyotype of species, out more, 2n = 25 (22A + X1X2Y). This number is very important for taxonomic and evolutionary inferences on the species, because of the 88 triatomine species with described karyotype, only T. rubrofasciata exhibits 25 chromosomes. Based on the hypothesis of the karyotype 2n = 22 (20A + XY) as ancestral for triatomines, we propose three evolutionary hypotheses for the emergence of the karyotype of T rubrofasciata, all supported by agmatoploidy events (fission). Basically the hypotheses are 1) fission for a pair of autosomes, resulting in 22 autosomes and later fission of sex chromosome X; 2) fission of pair of autosomes and the sex chromosome X concomitantly; 3) fission of sex chromosome X and subsequently fission of pair of autosomes. Thus, this study highlights for the first time the importance of the number of chromosomes of T. rubrofasciata as characteristic diagnosis in Triatominae subfamily and describes three evolutionary hypotheses that possibly led the emergence of karyotype of this insect of global importance.

Chromosomal characteristics and distribution of constitutive heterochromatin in the Matogrossensis and Rubrovaria subcomplexes.

Since 1966 the triatomines were grouped in complexes and specific subcomplexes. Although the complex and subcomplexes not have taxonomic importance, should be monophyletic groups and cytogenetic tools have proved to be of great importance to characterize these species groupings. Based on this, this paper aims to describe the chromosomal characteristics and heterochromatic pattern of Matogrossensis and Rubrovaria subcomplexes, in order to contribute to the taxonomic and evolutionary relationships of these vectors. In this study, at least three males from each species (Triatoma baratai, Triatoma costalimai, Triatoma guazu, Triatoma jurbergi, Triatoma matogrossensis, Triatoma vandae, Triatoma williami, Triatoma carcavalloi, Triatoma circummaculata, Triatoma klugi, Triatoma pintodiasi and Triatoma rubrovaria) were analyzed by means analyzed by means of cytogenetic techniques of C-banding. All species showed the same cytogenetic characteristics: 22 chromosomes, low variation in the size of autosomes, sex chromosome Y larger than X, initial prophase composed of only one heterochromatic chromocenter formed by the sex chromosomes X and Y (except for T. pintodiasi that presented the sex chromosomes individualized during all stages of prophase) and presence of constitutive heterochromatin restricted to sex chromosome Y. These characteristics, although common to Matogrossensis and Rubrovaria subcomplexes allow to distinguish these species of species grouped in most of South America subcomplexes, as Brasiliensis, Maculata, Sordida and Insfestans. Thus, the cytogenetic analysis was of extreme importance to differentiate both subcomplexes of the other subcomplexes of South America. However, probably due to evolutionary proximity existing between these subcomplexes was not possible to observar species differences that make up the Matogrossensis subcomplex of the Rubrovaria subcomplex. Therefore, we emphasize that new comparative analyzes, as experimental hybrid crosses and molecular cytogenetic analysis are necessary to clarify the evolutionary relationship between these important subcomplexes of vectors.

List of the type-specimens of Reduviidae (Hemiptera: Heteroptera) in the Triatomines Collection of the Oswaldo Cruz Institute, Brazil.

The Triatomines Collection of the Oswaldo Cruz Institute (CTIOC) holds 90 holotypes, seven syntypes, and 619 paratypes of the family Reduviidae, distributed in 44 genera and nine subfamilies: Chryxinae, Emesinae, Hammacerinae, Harpactorinae, Peiratinae, Reduviinae, Stenopodainae, Triatominae, and Vesciinae. A detailed list containing nomenclatural, taxonomic, and locality information about all these types is herein presented for the first time.

Trypanosoma cruzi Infection through the Oral Route Promotes a Severe Infection in Mice: New Disease Form from an Old Infection?

Oral transmission of Chagas disease has been documented in Latin American countries. Nevertheless, significant studies on the pathophysiology of this form of infection are largely lacking. The few studies investigating oral route infection disregard that inoculation in the oral cavity (Oral infection, OI) or by gavage (Gastrointestinal infection, GI) represent different infection routes, yet both show clear-cut parasitemia and heart parasitism during the acute infection. Herein, BALB/c mice were subjected to acute OI or GI infection using 5x10(4) culture-derived Trypanosoma cruzi trypomastigotes. OI mice displayed higher parasitemia and mortality rates than their GI counterparts. Heart histopathology showed larger areas of infiltration in the GI mice, whereas liver lesions were more severe in the OI animals, accompanied by higher Alanine Transaminase and Aspartate Transaminase serum contents. A differential cytokine pattern was also observed because OI mice presented higher pro-inflammatory cytokine (IFN-γ, TNF) serum levels than GI animals. Real-time PCR confirmed a higher TNF, IFN-γ, as well as IL-10 expression in the cardiac tissue from the OI group compared with GI. Conversely, TGF-ß and IL-17 serum levels were greater in the GI animals. Immunolabeling revealed macrophages as the main tissue source of TNF in infected mice. The high mortality rate observed in the OI mice paralleled the TNF serum rise, with its inhibition by an anti-TNF treatment. Moreover, differences in susceptibility between GI versus OI mice were more clearly related to the host response than to the effect of gastric pH on parasites, since infection in magnesium hydroxide-treated mice showed similar results. Overall, the present study provides conclusive evidence that the initial site of parasite entrance critically affects host immune response and disease outcome. In light of the occurrence of oral Chagas disease outbreaks, our results raise important implications in terms of the current view of the natural disease course and host-parasite relationship.

Phylogenetic relationships within the oliveirai complex (Hemiptera:Reduviidae:Triatominae).

The oliveirai complex is actually composed of nine related species of Triatominae that occur in the Pantanal ecosystem of Mato Grosso, Brazil. In order to confirm their specific status and infer their phylogenetic relationships, we performed a genetic and morphometric comparison concerning seven of the nine species of the complex. Isoenzyme analysis provided broadly similar phylogenetic information to that derived from discriminant analysis of size-free variables. The derived trees reveal two main species groups, one composed of three closed species (T. jurbergi, T. matogrossensis and T. vandae), and another one subdivided into two different pairs, the T. guazu-T. williami pair and the T. klugi-T. oliveirai pair. The results call the specific status of T. guazu and T. williami into question, support the possibility that the other analyzed species represent distinct taxa and query the existence of the oliveirai complex.

Synanthropic triatomines (Hemiptera, Reduviidae) in the state of Pernambuco, Brazil: geographical distribution and natural Trypanosoma infection rates between 2006 and 2007.

INTRODUCTION: The present study shows a descriptive analysis of triatomine occurrence and its natural Trypanosoma infection rates in the state of Pernambuco, Brazil, between 2006 and 2007. METHODS: Entomological data for the species, such as specimens captured in both intra and peridomiciles and natural infection index, were obtained via domiciliary capture in 147 municipalities from 11 Regional Managements of Health. The database was obtained from a sample of insects (100% infected and 20% non-infected) sent to the Central Laboratory of Pernambuco. RESULTS: A total of 18,029 triatomines were analyzed from 138 municipalities of the state. Triatoma pseudomaculata (35%), Triatoma brasiliensis (34%), and Panstrongylus lutzi (25%) were the most captured species. These species also showed a widespread geographical distribution in the state. Panstrongylus megistus, Triatoma petrocchiae, Triatoma melanocephala, Triatoma sordida, Rhodnius nasutus, Rhodnius neglectus, and Triatoma infestans showed more limited geographical distribution and lower relative abundance. The parasitological research showed that 8.8% of the triatomines were naturally infected with flagellates morphologically similar to Trypanosoma cruzi and 91.3% of them were captured inside houses in 113 municipalities. P. lutzi showed the highest rates of natural infection. CONCLUSIONS: After the control of T. infestans, synanthropic species, such as T. brasiliensis, T. pseudomaculata, and P. lutzi, maintain the risk of T. cruzi transmission to humans in the state of Pernambuco. These species are widely distributed, and infected specimens have been found inside houses. Thus, an enhanced surveillance and vector control of Chagas disease is recommended in Pernambuco.

Description of the feeding preferences of triatominae in the Chagas disease surveillance study for the State of Pernambuco, Brazil (Hemiptera: Reduviidae)

Abstract INTRODUCTION: Studying the feeding preferences of triatomines is an important entomological surveillance tool, since continuous surveillance of the disease is necessary. METHODS: The precipitin reaction was used to describe the feeding preferences of triatomines along with their natural infection by flagellates similar to Tyrpanosoma cruzi. Six hundred eighty-seven insects were examined, including Triatoma brasiliensis, Triatoma pseudomaculata, and Panstrongylus lutzi. RESULTS: Sixty-nine (10%) of 687 triatomines examined tested positive for flagellates similar to T. cruzi, and 8 (1.2%) of these fed on human blood. CONCLUSIONS: This study found potential transmitters of Chagas disease both inside and outside the domiciliar environment.

Registration of new geographical distribution of Panstrongylus geniculatus (Latreille) 1811 (Hemiptera, Reduviidae, Triatominae) in Brazil

The first report of the occurrence of Panstrongylus geniculatus (Latreille) 1811 was reported in the state of Pernambuco. Therefore its distribution was amplified, and now, such as P. megistus, is the most widely distributed species in Brazil. P. geniculatus was found by health workers in intradomiciliary environments in Vitória de Santo Antão, testing positive for flagellates observed by direct examination of intestinal contents, and Giemsa staining

Comparative study of the stridulatorium sulcus, buccula and rostrum of nymphs of Triatoma klugi Carcavallo et al, Triatoma vandae Carcavallo et al and Triatoma williami Galvão et al (Hemiptera: Reduviidae).

Ultrastructural analysis of the ventral region of the head--rostrum, buccula and stridulatorium sulcus--of 1st, 3rd and 5th instars of Triatoma klugi Carcavallo et al, Triatoma vandae Carcavallo et al, and Triatoma williami Galvão et al, are described in here. Morphological differences in the analyzed structures for all three Triatoma species studied were detected under scanning electron microscopy, allowing their grouping by their morphological similarities. Species-specific differences at each nymphal development stage were analyzed as well.