Ana C.A. Melo

Synthesis of vitellogenin by the follicle cells of Rhodnius prolixus

The synthesis and secretion of vitellogenin by the ovary of Rhodnius prolixus was investigated. Using whole ovary or epithelial cells isolated from follicles of different sizes, it is shown that the follicle cells are a site of synthesis for this protein in the ovary. The ovaries or follicle cells were incubated in vitro with [(35)S]-methionine or (32)Pi and the secretion of newly synthesized ovarian vitellogenin (O-Vg) was estimated by the radioactivity associated with the immunoprecipitate or acid-precipitate proteins in the culture medium. The radioactive O-Vg was analyzed by SDS-PAGE followed by autoradiography or after elution from a DEAE-Toyopearl column. The presence of O-Vg inside the follicle cells was detected by immunofluorescence and immunogold labels. Both methods revealed strong labeling inside the follicle cells. While the capacity for total protein synthesis by the follicle cells was maximal during the early phase of vitellogenesis (in small follicles), the synthesis of O-Vg reached its peak during the late phase of oocyte growth, just before formation of the chorion. A possible role for ovarian vitellogenin in Rhodnius and its relationship with Vg synthesis by the fat body is discussed.

A look inside odorant-binding proteins in insect chemoreception

Detection of chemical signals from the environment through olfaction is an indispensable mechanism for maintaining an insects life, evoking critical behavioral responses. Among several proteins involved in the olfactory perception process, the odorant binding protein (OBP) has been shown to be essential for a normally functioning olfactory system. This paper discusses the role of OBPs in insect chemoreception. Here, structural aspects, mechanisms of action and binding affinity of such proteins are reviewed, as well as their promising application as molecular targets for the development of new strategies for insect population management and other technological purposes.

Silencing the odorant receptor co-receptor RproOrco affects the physiology and behavior of the Chagas disease vector Rhodnius prolixus

Olfaction is one of the main sensory modalities that allow insects to interpret their environment. Several proteins, including odorant-binding proteins (OBPs) and odorant receptors (ORs), are involved in this process. Odorant receptors are ion channels formed by a binding unit OR and an odorant receptor co-receptor (Orco). The main goal of this study was to characterize the Orco gene of Rhodnius prolixus (RproOrco) and to infer its biological functions using gene silencing. The full-length RproOrco gene sequence was downloaded from VectorBase. This gene has 7 introns and is located in the genome SuperContig GL563069: 1,017,713-1,023,165. RproOrco encodes a protein of 473 amino acids, with predicted 7 transmembrane domains, and is highly expressed in the antennae during all R. prolixus developmental stages. The RNAi technique effectively silenced RproOrco, reducing the genes expression by approximately 73%. Interestingly, the effect of gene silencing persisted for more than 100 days, indicating a prolonged effect of dsRNA that was maintained even after molting. The phenotypic effects of silencing involved the following: (1) loss of the ability to find a vertebrate host in a timely manner, (2) decreased ingested blood volume, (3) delayed and decreased molt rate, (4) increased mortality rate, and (5) decreased egg laying. Our data strongly suggest that dsOrco disrupts R. prolixus host-finding behavior, which is further reflected in the blood ingestion, molting, mortality, and egg laying data. This study clearly demonstrates that Orco is an excellent target for controlling triatomine populations. Thus, the data presented here open new possibilities for the control of vector-borne diseases.

Chitin is a component of the Rhodnius prolixus midgut

Chitin is an essential component of the peritrophic matrix (PM), which is a structure that lines the insects gut and protects against mechanical damage and pathogens. Rhodnius prolixus (Hemiptera: Reduviidae) does not have a PM, but it has an analogous structure, the perimicrovillar membrane (PMM); chitin has not been described in this structure. Here, we show that chitin is present in the R. prolixus midgut using several techniques. The FTIR spectrum of the KOH-resistant putative chitin-material extracted from the midgut bolus showed peaks characteristic of the chitin molecule at 3500, 1675 and 1085 cm(1). Both the midgut bolus material and the standard chitin NMR spectra showed a peak at 1.88 ppm, which is certainly due to methyl protons in the acetamide a group. The percentages of radioactive N-acetylglucosamine (CPM) incorporated were 2 and 4% for the entire intestine and bolus, respectively. The KOH-resistant putative chitin-material was also extracted and purified from the N-acetylglucosamine radioactive bolus, and the radioactivity was estimated through liquid scintillation. The intestinal CHS cDNA translated sequence was the same as previously described for the R. prolixus cuticle and ovaries. Phenotypic alterations were observed in the midgut of females with a silenced CHS gene after a blood meal, such as retarded blood meal digestion; the presence of fresh blood that remained red nine days after the blood meal; and reduced trachea and hemozoin content compared with the control. Wheat germ agglutinin (a specific probe that detects chitin) labeling proximal to the intestine (crop and midgut) was much lower in females with a silenced CHS gene, especially in the midgut region, where almost no fluorescence signal was detected compared with the control groups. Midguts from females with a CHS gene silenced by dsRNA-CHS and control midguts pre-treated with chitinase showed that the chitin-derived fluorescence signal decreased in the region around the epithelium, the region facing the midgut and projections towards the intestinal lumen when evaluated microscopically. The relative reduction in CHS transcripts by approximately 80% using an RNAi assay supports the phenotypical alterations in the midgut observed using fluorescence microscopy assays. These data show that chitin is present in the R. prolixus midgut epithelium and in its surface projections facing the lumen. The CHS gene expression and the presence of chitin in the R. prolixus midgut may suggest a target for controlling Chagas disease vectors and addressing this public health problem.

Comparative proteome analysis reveals that blood and sugar meals induce differential protein expression in Aedes aegypti female heads

Aedes aegypti females ingest sugar or blood to obtain the nutrients needed to maintain cellular homeostasis. During human blood ingestion, female mosquitoes may transmit different viruses such as dengue, yellow fever and, more recently, zika and chikungunya. Here, we report changes in protein expression in the heads of adult female Ae. aegypti mosquitoes in response to the ingestion of blood or sugar. Proteins extracted from the heads of Ae. aegypti fed exclusively on blood (BF) or sugar (SF) were trypsin hydrolyzed (off‐gel) and analyzed by the reverse‐phase nano‐liquid chromatography coupled with hybrid mass spectrometry. A total of 1139 proteins were identified in female heads, representing 7.4% of the predicted proteins in Ae. aegypti genome (total = 15 419 active genes). Gene ontology annotation and categories showed that, in this insect, the head was rich in proteins involved in the metabolic process, proton transport, organelle, macromolecular complex, structural molecule activity, antioxidant activity, and catalytic activity. Our report is the first indicating that many of the annotated genes are translated into functional proteins in heads of adult female Ae. aegypti. Interestingly, we identified 8.7 times more exclusively expressed proteins involved in signal transduction, replication–transcription–translation (5.5 x), and transport (2.9 x) activity in BF than in SF groups. This paper discusses the protein profile of Ae. aegypti female heads and its implications for blood ingestion and carbohydrate intake.

History, epidemiology and diagnostics of dengue in the American and Brazilian contexts: a review

Dengue virus (DENV), an arbovirus transmitted by mosquitoes, has become a major threat to American human life, reaching approximately 23 million cases from 1980 to 2017. Brazil is among the countries most affected by this terrible viral disease, with 13.6 million cases. DENV has four different serotypes, DENV1-4, which show a broad clinical spectrum. Dengue creates a staggering epidemiological and economic burden for endemic countries. Without a specific therapy and with a commercial vaccine that presents some problems relative to its full effectiveness, initiatives to improve vector control strategies, early disease diagnostics and the development of vaccines and antiviral drugs are priorities. In this study, we present the probable origins of dengue in America and the trajectories of its spread. Overall, dengue diagnostics are costly, making the monitoring of dengue epidemiology more difficult and affecting physicians’ therapeutic decisions regarding dengue patients, especially in developing countries. This review also highlights some recent and important findings regarding dengue in Brazil and the Americas. We also summarize the existing DENV polymerase chain reaction (PCR) diagnostic tests to provide an improved reference since these tests are useful and accurate at discriminating DENV from other flaviviruses that co-circulate in the Americas. Additionally, these DENV PCR assays ensure virus serotyping, enabling epidemiologic monitoring.

Reverse chemical ecology-based approach leading to the accidental discovery of repellents for Rhodnius prolixus, a vector of Chagas diseases refractory to DEET

Rhodnius prolixus is one of the most important vectors of Chagas disease in Central and South America for which repellents and attractants are sorely needed. Repellents like DEET, picaridin, and IR3535 are widely used as the first line of defense against mosquitoes and other vectors, but they are ineffective against R. prolixus. Our initial goal was to identify in R. prolixus genome odorant receptors sensitive to putative sex pheromones. We compared gene expression of 21 ORs in the R. prolixus genome, identified 4 ORs enriched in male (compared with female) antennae. Attempts to de-orphanize these ORs using the Xenopus oocyte recording system showed that none of them responded to putative sex pheromone constituents. One of the them, RproOR80, was sensitive to 4 compounds in our panel of 109 odorants, namely, 2-heptanone, γ-octalactone, acetophenone, and 4-methylcychohexanol. Interestingly, these compounds, particularly 4-methylcyclohexanol, showed strong repellency activity as indicated not only by a significant decrease in residence time close to a host, but also by a remarkable reduction in blood intake. 4-Methylcyclohexanol-elicited repellency activity was abolished in RNAi-treated insects. In summary, our search for pheromone receptors led to the discovery of repellents for R. prolixus.

Functional Characterization of Odorant Binding Protein 27 (RproOBP27) From Rhodnius prolixus Antennae

Olfactory proteins mediate a wide range of essential behaviors for insect survival. Odorant binding proteins (OBPs) are small soluble olfactory proteins involved in the transport of odor molecules (=odorants) through the sensillum lymph to odorant receptors, which are housed on the dendritic membrane of olfactory sensory neurons also known as olfactory receptor neurons. Thus, a better understanding of the role(s) of OBPs from Rhodnius prolixus, one of the main vectors of Chagas disease, may ultimately lead to new strategies for vector management. Here we aimed at functionally characterize OBPs from R. prolixus. Genes of interest were selected using conventional bioinformatics approaches and subsequent quantification by qPCR. We screened and estimated expression in different tissues of 17 OBPs from R. prolixus adults. These analyses showed that 11 OBPs were expressed in all tissues, whereas six OBP genes were specific to antennae. Two OBP genes, RproOBP6 and RproOBP13, were expressed in both male and female antennae thus suggesting that they might be involved in the recognition of semiochemicals mediating behaviors common to both sexes, such host finding (for a blood meal). Transcripts for RproOBP17 and RproOBP21 were enriched in female antennae and possibly involved in the detection of oviposition attractants or other semiochemicals mediating female-specific behaviors. By contrast, RproOBP26 and RproOBP27 might be involved in the reception of sex pheromones given that their transcripts were highly expressed in male antennae. To test this hypothesis, we silenced RproOBP27 using RNAi and examined the sexual behavior of the phenotype. Indeed, adult males treated with dsOBP27 spent significantly less time close to females as compared to controls. Additionally, docking analysis suggested that RproOBP27 binds to putative sex pheromones. We therefore concluded that RproOBP27 might be a pheromone-binding protein.

Otologic Surgery Preoperative Imaging: Mandatory or Recommended? Useful or Defensive?

Objectives: Look for and implement guidelines for preoperative imaging requests to facilitate clinical practice and avoid medico-legal problems. Methods: A review of contemporary English medical literature via Medline using the terms imaging, preoperative imaging, computed tomography (CT) scan/CT, magnetic resonance imaging (MRI)/magnetic resonance plus otosclerosis, chronic otitis media, cholesteatoma, simple chronic otitis, tympanoplasty/myringoplasty, and middle ear was performed. Abstracts were reviewed independently by two authors and relevant articles were then evaluated. Indications for preoperative imaging study for each pathology considered, benefits, main findings and the cost-beneficial relationship were investigated. Results: Few articles address this issue. Otosclerosis: 152 articles, with some of the studies referring to the assessment of activity and extension of the disease, but only 3 were related to the demand for imaging. Myringoplasty/tympanoplasty: a total of 87 items were found but only 4 address the issue of preoperative imaging evaluation. Chronic otitis media/cholesteatoma: 467 articles, with 32 approaching this subject. For each abstract evaluated, conditions were considered: the indications and relevant findings versus the diagnosis controversial aspects and the real need of the request (findings that can be easily diagnosed intraoperatively). Conclusions: There is a growing number of otolaryngologists who routinely request preoperative imaging before middle ear surgery based on ambiguous criteria. The proposal for the creation of guidelines will help clinicians in their diagnostic decisions and to avoid medico-legal problems.