An Integrative Sialomic Analysis Reveals Molecules From Triatoma sordida (Hemiptera: Reduviidae).

Triatomines have evolved salivary glands that produce versatile molecules with various biological functions, including those leading their interactions with vertebrate hosts' hemostatic and immunological systems. Here, using high-throughput transcriptomics and proteomics, we report the first sialome study on the synanthropic triatomine Triatoma sordida. As a result, 57,645,372 reads were assembled into 26,670 coding sequences (CDS). From these, a total of 16,683 were successfully annotated. The sialotranscriptomic profile shows Lipocalin as the most abundant protein family within putative secreted transcripts. Trialysins and Kazal-type protease inhibitors have high transcript levels followed by ubiquitous protein families and enzyme classes. Interestingly, abundant trialysin and Kazal-type members are highlighted in this triatomine sialotranscriptome. Furthermore, we identified 132 proteins in T. sordida salivary gland soluble extract through LC-MS/MS spectrometry. Lipocalins, Hemiptera specific families, CRISP/Antigen-5 and Kazal-type protein inhibitors proteins were identified. Our study provides a comprehensive description of the transcript and protein compositions of the salivary glands of T. sordida. It significantly enhances the information in the Triatominae sialome databanks reported so far, improving the understanding of the vector's biology, the hematophagous behaviour, and the Triatominae subfamily's evolution.

Exploring the molecular complexity of Triatoma dimidiata sialome.

Triatoma dimidiata, a Chagas disease vector widely distributed along Central America, has great capability for domestic adaptation as the majority of specimens caught inside human dwellings or in peridomestic areas fed human blood. Exploring the salivary compounds that overcome host haemostatic and immune responses is of great scientific interest. Here, we provide a deeper insight into its salivary gland molecules. We used high-throughput RNA sequencing to examine in depth the T. dimidiata salivary gland transcriptome. From >51 million reads assembled, 92.21% are related to putative secreted proteins. Lipocalin is the most abundant gene family, confirming it is an expanded family in Triatoma genus salivary repertoire. Other putatively secreted members include phosphatases, odorant binding protein, hemolysin, proteases, protease inhibitors, antigen-5 and antimicrobial peptides. This work expands the previous set of functionally annotated sequences from T. dimidiata salivary glands available in NCBI from 388 to 3815. Additionally, we complemented the salivary analysis through proteomics (available data via ProteomeXchange with identifier PXD008510), disclosing the set complexity of 119 secreted proteins and validating the transcriptomic results. Our large-scale approach enriches the pharmacologically active molecules database and improves our knowledge about the complexity of salivary compounds from haematophagous vectors and their biological interactions. SIGNIFICANCE: Several haematophagous triatomine species can transmit Trypanosoma cruzi, the etiological agent of Chagas disease. Due to the reemergence of this disease, new drugs for its prevention and treatment are considered priorities. For this reason, the knowledge of vector saliva emerges as relevant biological finding, contributing to the design of different strategies for vector control and disease transmission. Here we report the transcriptomic and proteomic compositions of the salivary glands (sialome) of the reduviid bug Triatoma dimidiata, a relevant Chagas disease vector in Central America. Our results are robust and disclosed unprecedented insights into the notable diversity of its salivary glands content, revealing relevant anti-haemostatic salivary gene families. Our work expands almost ten times the previous set of functionally annotated sequences from T. dimidiata salivary glands available in NCBI. Moreover, using an integrated transcriptomic and proteomic approach, we showed a correlation pattern of transcription and translation processes for the main gene families found, an important contribution to the research of triatomine sialomes. Furthermore, data generated here reinforces the secreted proteins encountered can greatly contribute for haematophagic habit, Trypanosoma cruzi transmission and development of therapeutic agent studies.

A Deep Insight into the Sialome of Rhodnius neglectus, a Vector of Chagas Disease.

BACKGROUND: Triatomines are hematophagous insects that act as vectors of Chagas disease. Rhodnius neglectus is one of these kissing bugs found, contributing to the transmission of this American trypanosomiasis. The saliva of hematophagous arthropods contains bioactive molecules responsible for counteracting host haemostatic, inflammatory, and immune responses. METHODS/PRINCIPAL FINDINGS: Next generation sequencing and mass spectrometry-based protein identification were performed to investigate the content of triatomine R. neglectus saliva. We deposited 4,230 coding DNA sequences (CDS) in GenBank. A set of 636 CDS of proteins of putative secretory nature was extracted from the assembled reads, 73 of them confirmed by proteomic analysis. The sialome of R. neglectus was characterized and serine protease transcripts detected. The presence of ubiquitous protein families was revealed, including lipocalins, serine protease inhibitors, and antigen-5. Metalloproteases, disintegrins, and odorant binding protein families were less abundant. CONCLUSIONS/SIGNIFICANCE: The data presented improve our understanding of hematophagous arthropod sialomes, and aid in understanding hematophagy and the complex interplay among vectors and their vertebrate hosts.

[Ultrastructural alterations in larvae of Aedes aegypti subject to labdane diterpene isolated from Copaifera reticulata (Leguminosae) and a fraction enriched with tannins of Magonia pubescens (Sapindaceae)]. / Alterações ultraestruturais em larvas de Aedes aegypti submetidas ao diterpeno labdano, isolado de Copaifera reticulata (Leguminosae), e à uma fração rica em taninos de Magonia pubescens (Sapindaceae).

INTRODUCTION: Dengue is an important public health problem in many countries and its main vector Aedes aegypti, is the mosquito most adapted to urban areas. For the first time, the mechanism of action of labdane diterpenoid extracted from Copaifera reticulata and the fraction enriched of catechin tannins extracted from Magonia pubescens is demonstrated through ultrastructural alterations of Aedes aegypti larvae. METHODS: Experiments were performed using a 0.9 ppm solution of diterpenoid and 3.7 ppm of the fraction as the main catechin tannin of molecular mass 846 Da. The compounds were obtained by thin layer chromatography and identified by nuclear magnetic resonance of hydrogen and mass spectrometry. Larvae that achieved lethargic state were collected and dissected. Next, they were contrasted with 1% uranyl acetate, dehydrated, embedded and polymerized. Ultrathin sections were made, mixed with 3% uranyl acetate and lead citrate and placed in an electron microscope. RESULTS: The main ultrastructural alterations caused by the diterpenoid and by tanins in larvae of Aedes aegypti were: cytoplasmic vacuolation, alteration of microvilli, cellular aging, cell disruption and degeneration, formation of secretion vesicles and structural changes in microvilli, irregular nuclei and displacement of cells in the basal lamina. CONCLUSIONS: The fraction containing tannins and the diterpenoid caused the death of Aedes aegypti larvae by cell destruction in the midgut.

Larvicidal activity of oil-resin fractions from the Brazilian medicinal plant Copaifera reticulata Ducke (Leguminosae-Caesalpinoideae) against Aedes aegypti (Diptera, Culicidae).

Oil-resin fractions from Copaifera reticulata Ducke (Leguminosae-Caesalpinoideae) were evaluated for larvicidal activity on third larval instars of Aedes aegypti, in searching for alternative control methods for this mosquito. The bioactive fractions were chemically monitored by thin-layer chromatography, (1)H and 13C nuclear magnetic resonance and mass spectrometry. Bioassays were performed using five repetitions, at a temperature of 28+/-1 degrees C, relative humidity of 80+/- 5% and light and dark cycles of 12h. Mortality was indicated by darkening of the cephalic capsule after 24h of exposure of the larvae to the solutions. The most active fractions were CRM1-4 (sesquiterpenes) and CRM5-7 (labdane diterpenes), which showed LC50 values of 0.2 and 0.8 ppm, respectively.

[Geographical distribution and indicators entomologic of sinantropic triatomines captured in the State of Goiás]. / Distribuição geográfica e indicadores entomológicos de triatomíneos sinantrópicos capturados no Estado de Goiás.

The geographic distribution of triatomines in Goiás and entomological indicators in the home environment are presented: home infestation, home triatomine density and vector infection by Trypanosoma cruzi. The indicators studied were from triatomines captured inside and outside homes in 201 municipalities in the State of Goiás, Brazil, between 2000 and 2003. A total of 249,868 home units were investigated and 51,570 triatomines were captured, and 335 specimens were found to be infected with Trypanosoma cruzi. Infestation outside the home was significantly greater than infestation inside the home for the species Triatoma sordida, followed by Panstrongylus megistus. The contrary was observed with the species Rhodnius neglectus, Panstrongylus geniculatus and Triatoma pseudomaculata (p<0.018). There was no significant difference between infestations inside and outside the home for the species Panstrongylus diasi, Triatoma costalimai and Triatoma williami. Only one specimen of Triatoma infestans was captured in the year 2000.

Specific treatment for Trypanosoma cruzi: lack of efficacy of allopurinol in the human chronic phase of Chagas disease.

Thirty-five individuals from endemic areas of Central Brazil (age range, 18-64 years; 19 women) in the chronic phase of Chagas disease, with positive serology and presence of circulating parasites detected by one or more recent positive xenodiagnosis, were selected for this study. Allopurinol (900 mg/d) or placebo was administered in a double-blind clinical trial for 60 days. After codes were broken, 23 had been allocated to the intervention group and 12 to the placebo group. Side effects were observed in 11 patients in the intervention group and in 1 in the placebo group. Seventeen patients in the intervention group and 10 in the placebo group completed the trial. Follow-up was performed by monthly xenodiagnosis and serologic tests every 3 months during the first year and at the end of the trial. Xenodiagnosis remained positive in all 17 of the treated group and in all 10 of the placebo group. Serologic tests were persistently positive in both groups after treatment. We concluded that, at the doses used, allopurinol was not effective to clear, in our region, Trypanosoma cruzi from peripheral blood of infected individuals.

[Evaluation of the inhibiting activity of the diflubenzuron on the ecdysis of larvae of Aedes aegypti (Linnaeus, 1762) (Diptera, Culicidae)]. / Avaliação da atividade inibidora do diflubenzuron na ecdise das larvas de Aedes aegypti (Linnaeus, 1762) (Diptera, Culicidae).

The inhibiting activity of diflubenzuron on the ecdysis of Aedes aegypti larvae was evaluated, with a view to using this product in mosquito control. This study also aimed to determine the interaction between this product, the type of artificial containers and the susceptibility of the mosquito. Bioassays were carried out in the backyard of a residence, using seven kinds of artificial habitats: tires, glass, concrete roofing, cans, plastic containers, cement and pottery. In each kind of artificial habitat, 20 Aedes aegypti larvae in the 4th instar were set. The same number of larvae was used as a control. Each test was repeated five times and the observation of mortality was done once every 24 hours, until 100% mortality was reached at 1 ppm. There was no significant difference between the main surveillance periods of the larvae, nor between the various kinds of artificial habitats. A significant difference was found between the instars, in that the 3rd instar was the most resistant to diflubenzuron inhibiting activity. It was also shown that concentrations did not interact with instars or material of the artificial habitats at the 5% significance level.

[Toxicity of the ethanol extract of Magonia pubescens on larvae Aedes aegypti]. / Toxicidade do extrato etanólico de Magonia pubescens sobre larvas de Aedes aegypti.

Crude ethanol extracted from Magonia pubescens trunks was utilized to prove its effect in the intestinal tract of Aedes aegypti 3rd instar larvae. Exposure times (2, 4, 6, 8, 10, 12, and 13 hours) were tested to verify when the morphological alterations begin to occur, in the intestinal tract of the larvae. The toxic effect of the extract was mainly in the midgut, beginning at the anterior midgut and followed through to posterior midgut. The main alterations observed were partial or total cell destruction, high citoplasmatic vacuolization, increase of subperitrophic space, cell hypertrophy and the epithelium did not maintain its monolayer appearance. The alterations began after four hours of exposure to M. pubescens extract. Observations of tissue sections from larvae treated for different lengths of time revealed a wide variation in the degree of damage between exposure periods and midgut larvae region. The present study provides evidence regarding the mode of action of the M. pubescens extract and suggests its potential utilization as a larvicide to control Aedes aegypti mosquito.