- Molecular Biology of Parasites & Disease Vectors
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- The Molecular Biology of Insect Disease Vectors - A Methods Manual | Julian Crampton | Springer
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Previous results indicate that the expression level of TcSMUG L -encoded products is quite variable among epimastigotes from different T. Therefore, as a first step toward the validation of our R. The results were normalized by re-probing the membrane with antiserum directed against TcGDH. These products were removed from the parasite surface following PI-PLC treatment  , a molecular signature of GPI-anchored molecules not shown , and were specifically retained following ConA chromatography Fig.
As shown in Fig. A minor fraction was also revealed in the pellet, which might be ascribed to incomplete extraction.
Molecular Biology of Parasites & Disease Vectors
Together, these results strongly suggest that Dm28c epimastigotes express high levels of fully processed TcSMUG L product on their surface. C Butanol extraction analysis of Dm28c delipidated epimastigotes. Molecular mass markers in kDa are indicated at right. As a first set of experiments, in posterior R. Similar adhesion rates In contrast, attachment of only A dose-dependent effect on the ex vivo attachment of epimastigotes was verified for the latter molecules, indicating that the presence of either synthetic peptide blocked a potential ligand-receptor interaction involved in epimastigote attachment Fig.
Similarly, midgut incubation with 0.
Therefore, our results showed that the pre-incubation of R. Midguts obtained from male fifth-instar nymphs 10 days after the bloodmeal were previously incubated for 30 min in PBS supplemented with the indicated mucin peptides and added with BHI interaction medium containing flagellates 2. Pre-incubation with mucin peptides was omitted in control non-treated group. Adhered epimastigotes were counted per epithelial cells in 10 different fields of each midgut preparation. B Pre-incubation in 0. C Pre-incubation in 0.
Asterisk represents experimental groups with statistical significance compared to the control.
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In the control group, the infection levels varied from 3. Direct counts revealed 2. Light microscopy of R. Toluidine-stained granules were observed in the apical and medial region, where a round nucleus was located. As previously described  , these epithelial cells were closely joined at their medial and basal regions, whereas a brush border associated with the PMM was observed at the luminal surface of their apical regions Fig.
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No significant labeling was obtained after incubation of R. However, in line with previous results, fluorescence of specific binding sites was observed on the surface of luminal posterior midgut cells after pre-incubation with biotin-labeled TcSMUG L Fig.
Unexpectedly, the samples pre-incubated with TSSA also showed some intracellular staining, particularly in the nucleolus, which may be attributed to partial permeabilization of the cells during fixation. A Light microscopy showing single-globe columnar epithelial cells white star and PMM white arrow. B Fluorescence microscopy showing that no demarcation was observed after incubation with avidin-FITC-labeled conjugate alone. Fluorescence of the surface and nucleolus of the midgut cells is indicated by white and black arrows respectively. During its life cycle, T.
Depending on the parasite developmental stage and the nature of the involved molecules, these interactions trigger a variety of events such as bidirectional cell signaling, host cell internalization, parasite replication or transformation to infective stages  , . Within the triatomid vector, different lines of research have established that molecules able to inhibit parasite attachment to insect tissues ex vivo also often efficiently block the in vivo development of T.
For instance, purified GIPLs were shown to bind to the luminal surface of the posterior midgut. Accordingly, their exogenous addition dramatically impaired both ex vivo attachment of epimastigotes to this organ and the flagellate multiplication in the insect digestive tract, which prevented the successful colonization of the vector . Similar effects were described for different carbohydrate-binding proteins CBPs of the epimastigote surface with a strong affinity for higher glycan oligomers and sulfated glycosaminoglycans S-GAGs present in the posterior midgut of R.
The net negative charge of both S-GAGs and specific carbohydrates may act as a first, non-specific step prior to T. In addition, an antiserum raised against R. The entire surface, including the cell body and the flagellum, of various T. This N-terminal peptide, which is not predicted to be O -glycosylated, is thus ideally suited to participate in cell-to-cell interaction phenomena .
The results presented here strongly suggest that the N-terminal peptide of TcSMUG L products is required for efficient interaction between the parasite and the insect midgut and the subsequent growth of the flagellate in the invertebrate host. As shown, addition of the exogenous peptide led to a significant reduction in ex vivo adhesion to the insect midgut, and also inhibition of in vivo development within vectors.
Due to its small molecular size, this effect is unlikely to be caused by steric effects, where the TcSMUG L peptide would prevent access of parasite recognition molecules to specific sites in the insect gut cells. Quite the opposite, we favor the hypothesis that the exogenous TcSMUG L peptide exerts its inhibitory effect by outcompeting the parasite binding sites in the triatomine luminal surface of the midgut epithelium.
This idea is further supported by histochemical data showing intense labeling of the surface of luminal posterior midgut cells after pre-incubation with biotin-labeled TcSMUG L peptide. Therefore, it is likely that TcSMUG L products act as surface adhesion molecules, promoting epimastigote adhesion and colonization through recognition of specific receptor s on insect cells.
In this framework, a distinct expression profile verified for TcSMUG L products  could contribute to the biological heterogeneity found between different isolates of T.
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Moreover, drastic reduction in TcSMUG L expression upon differentiation to metacyclic trypomastigotes suggests a developmental regulation program that could help to explain why these latter forms are detached from the midgut surface . One unexpected and puzzling finding was that the exogenous TSSA-derived peptide showed adhesion properties to insect midgut cells, as well as ex vivo inhibition on epimastigote attachment. It is worth mentioning that TSSA belongs to the TcMUC group of genes, which is expressed during the mammalian-dwelling stages of the protozoan  ,  , .
In particular, TSSA expression is restricted to the surface of blood trypomastigotes, the parasite stage ingested by the vector during an infective blood meal, and amastigote-to-bloodstream trypomastigote intermediate forms. From a structural staindpoint, and despite showing similar bias in amino acid composition with Cys, Phe, Trp and Tyr amino acids -all residues that could perturb the physicochemical properties of T.
Indeed, the labeling pattern obtained for TSSA in posterior midgut sections is different than that obtained for the TcSMUG L peptide, suggesting they recognize different receptor s on the surface of insect cells, although more studies would be required to address this point. Importantly, and in strict correlation with its expression profiling, the interaction between TSSA and insect midgut cells seems to have no biological relevance, as it had no effect on parasite in vivo development. Although little is known about the mechanisms leading to the remodeling of the surface coat when the flagellate moves from the mammal into the insect vector, it is reasonable to suppose that TSSA is shed during this process.
Free in the insect stomach, TSSA may reach the posterior midgut and be recognized by PMM receptors for mucins or other glycoconjugates. Transfer of antigenic epitopes from T. Indeed, our results demonstrate that these products are involved in successful adhesion to the epithelial cells of insect vectors both ex vivo and in vivo , although the exact molecular mechanism, and particularly the putative receptor on the surface of the insect cells, should be further explored. Most importantly, a severe reduction in flagellate population in the digestive tract of R.
Light microscopy of toluidin blue-stained posterior midgut cells of R. Oblique a and transverse b sections of the apical region of columnar epithelial cells, with brush border associated with perimicrovillar membranes thick black arrow , round nuclei thin black arrow and the posterior midgut lumen L.
The Molecular Biology of Insect Disease Vectors - A Methods Manual | Julian Crampton | Springer
We also thank Dr. Abstract Background TcSMUG L products were recently identified as novel mucin-type glycoconjugates restricted to the surface of insect-dwelling epimastigote forms of Trypanosoma cruzi , the etiological agent of Chagas disease. Methodology and Principal Findings Here, we investigated the putative roles of TcSMUG L mucins in both in vivo development and ex vivo attachment of epimastigotes to the luminal surface of the digestive tract of Rhodnius prolixus.
Author Summary Chagas disease, the major tropical human disease in much of Latin America, affects approximately 11 million people. Materials and Methods Insects and Parasites R.
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Ethics Statement R. Peptides Peptides used in this study were synthesized bearing an acetyl group on their N-termini and a C-terminal Cys residue GenScript. Ex Vivo Interaction between R. Light Microscopy Posterior midgut compartments obtained by dissection were fixed for 2 h at room temperature in 2. Download: PPT. Figure 1. Figure 2. Effect of surface mucins on ex vivo T.
Figure 3. Effect of surface mucins on T. Figure 4. Photomicrographs of posterior midgut epithelial cells of fifth-instar R. Discussion During its life cycle, T. Supporting Information. Figure S1. References 1. Estudos Sobre a Morfologia e o ciclo Evolutivo do Schizotrypanum cruzi n. View Article Google Scholar 2. Mem Inst Oswaldo Cruz 3: — View Article Google Scholar 3. Accessed 06 April J Insect Phys 11— View Article Google Scholar 5. Advances in Insect Physiology. Trends in Parasitology — View Article Google Scholar 7.
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Dengue and dengue hemorrhagic fever are increasing public health problems with an estimated million new infections each year. A "sterile-male-release" strategy based on the release of mosquitoes carrying a conditional dominant lethal gene is an attractive new control methodology. These strains eliminate the need for sterilization by irradiation, permit male-only release "genetic sexing" , and enable the release of eggs instead of adults. The snippet could not be located in the article text. This may be because the snippet appears in a figure legend, contains special characters or spans different sections of the article.
Published online Feb PMID: Guoliang Fu , a, b, 1 Rosemary S. James , c, d, 2 and Luke Alphey a, b, 2.