[Show abstract][Hide abstract] ABSTRACT: The triatomine, Rhodnius prolixus, is a major vector of Trypanosoma cruzi, the causative agent of Chagas disease in Latin America. It has a strictly blood-sucking habit in all life stages, ingesting large amounts of blood from vertebrate hosts from which it can acquire pathogenic microorganisms. In this context, the production of antimicrobial peptides (AMPs) in the midgut of the insect is vital to control possible infection, and to maintain the microbiota already present in the digestive tract.
[Show abstract][Hide abstract] ABSTRACT: Except for honey as food, and silk for clothing and pollination of plants, people give little thought to the benefits of insects in their lives. This overview briefly describes significant recent advances in developing insect natural products as potential new medicinal drugs. This is an exciting and rapidly expanding new field since insects are hugely variable and have utilised an enormous range of natural products to survive environmental perturbations for 100s of millions of years. There is thus a treasure chest of untapped resources waiting to be discovered. Insects products, such as silk and honey, have already been utilised for thousands of years, and extracts of insects have been produced for use in Folk Medicine around the world, but only with the development of modern molecular and biochemical techniques has it become feasible to manipulate and bioengineer insect natural products into modern medicines. Utilising knowledge gleaned from Insect Folk Medicines, this review describes modern research into bioengineering honey and venom from bees, silk, cantharidin, antimicrobial peptides, and maggot secretions and anticoagulants from blood-sucking insects into medicines. Problems and solutions encountered in these endeavours are described and indicate that the future is bright for new insect derived pharmaceuticals treatments and medicines.
Evidence-based Complementary and Alternative Medicine 05/2014; 2014:904958. · 2.18 Impact Factor
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[Show abstract][Hide abstract] ABSTRACT: Using specific oligonucleotides, 5'- and 3'-RACE and sequencing, two cDNAs encoding serine carboxypeptidases (tbscp-1 and tbscp-2) from the midgut of the blood sucking heteropteran Triatoma brasiliensis were identified. Both cDNAs with an open reading frame of 1389 bp, encode serine carboxypeptidase precursors of 463 amino acid residues, which possess a signal peptide cleavage site after Ala19. Analysis of tbscp-1 and tbscp-2 genomic DNA showed an absence of introns in both sequences and the presence of a further intron-free SCP encoding gene (tbscp-2b). By reverse transcription polymerase chain reaction (RT-PCR), tbscp-1 and tbscp-2 transcript abundance was found similarly in fifth instar nymphs at different days after feeding (daf), high in the posterior midgut (small intestine), lower in the anterior midgut (stomach) and fat body and almost undetectable in the salivary glands. In the anterior, middle and posterior regions of the small intestine at 5 daf the transcript abundance of both genes was almost identical. Also in adult female and male insects at 5 daf both genes showed the strongest signal in the posterior midgut. Molecular modeling suggested that TBSCP-1 has carboxypeptidase D activity; activities against Hippuryl-Phenylalanine and Hippuryl-Arginine were also located at the posterior midgut, both were induced after blood feeding. Treatment of the posterior midgut extracts with the serine protease inhibitor PMSF strongly reduced carboxypeptidase activity. These findings suggest that triatomines might use serine carboxypeptidases, which are usually found in lysosomes, as digestive enzymes in the posterior midgut lumen, from which TBSCP-1 and TBSCP-2 are possible candidates to fulfill this function.
Journal of insect physiology 02/2014; · 2.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have used phase-contrast X-ray microtomography (PPC-μCT) to study
the head of the blood-feeding bug, Rhodnius prolixus, which is one of
the most important insect vector of Trypanosoma cruzi, ethiologic agent
of Chagas disease in Latin America. Images reconstructed from
phase-retrieved projections processed by ANKA phase are compared to
those obtained through direct tomographic reconstruction of the
flat-field-corrected transmission radiographs. It should be noted that
the relative locations of the important morphological internal
structures are observable with a precision that is difficult to obtain
without the phase retrieval approach.
Radiation Physics and Chemistry 02/2014; · 1.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chagas disease kills 2.5 thousand people per year of 15 million persons infected in Latin America. The disease is caused by the protozoan, Trypanosome cruzi, and vectored by triatomine insects, including Panstrongylus megistus, an important vector in Brazil. Medicines treating Chagas disease have unpleasant side effects and may be ineffective, therefore, alternative control techniques are required. Knowledge of the T. cruzi interactions with the triatomine host needs extending and new targets/strategies for control identified. Serine and cysteine peptidases play vital roles in protozoan life cycles including invasion and entry of T. cruzi into host cells. Peptidase inhibitors are, therefore, promising targets for disease control.
SDS PAGE and chromatograpy detected and isolated a P. megistus serpin which was peptide sequenced by mass spectrometry. A full amino acid sequence was obtained from the cDNA and compared with other insect serpins. Reverse transcription PCR analysis measured serpin transcripts of P. megistus tissues with and without T. cruzi infection. Serpin homology modeling used the Swiss Model and Swiss-PDB viewer programmes.
The P. megistus serpin (PMSRP1) has a ca. 40 kDa molecular mass with 404 amino acid residues. A reactive site loop contains a highly conserved hinge region but, based on sequence alignment, the normal cleavage site for serine proteases at P1-P1[prime] was translocated to the putative position P4[prime]-P5. A small peptide obtained corresponded to the C-terminal 40 amino acid region. The secondary structure of PMSRP1 indicated nine alpha-helices and three beta-sheets, similar to other serpins. PMSRP1 transcripts occurred in all tested tissues but were highest in the fat body and hemocytes. Levels of mRNA encoding PMSRP1 were significantly modulated in the hemocytes and stomach by T. cruzi infection indicating a role for PMSRP1 in the parasite interactions with P. megistus.
For the first time, a constitutively expressed serpin has been characterized from the hemolymph of a triatomine. This opens up new research avenues into the roles of serine peptidases in the T. cruzi/P. megistus association. Initial experiments indicate a role for PMSRP1 in T. cruzi interactions with P. megistus and will lead to further functional studies of this molecule.
[Show abstract][Hide abstract] ABSTRACT: The bloodsucking hemipteran Rhodnius prolixus is a vector of Chagas' disease, which affects 7-8 million people today in Latin America. In contrast to other hematophagous insects, the triatomine gut is compartmentalized into three segments that perform different functions during blood digestion. Here we report analysis of transcriptomes for each of the segments using pyrosequencing technology. Comparison of transcript frequency in digestive libraries with a whole-body library was used to evaluate expression levels. All classes of digestive enzymes were highly expressed, with a predominance of cysteine and aspartic proteinases, the latter showing a significant expansion through gene duplication. Although no protein digestion is known to occur in the anterior midgut (AM), protease transcripts were found, suggesting secretion as pro-enzymes, being possibly activated in the posterior midgut (PM). As expected, genes related to cytoskeleton, protein synthesis apparatus, protein traffic, and secretion were abundantly transcribed. Despite the absence of a chitinous peritrophic membrane in hemipterans - which have instead a lipidic perimicrovillar membrane lining over midgut epithelia - several gut-specific peritrophin transcripts were found, suggesting that these proteins perform functions other than being a structural component of the peritrophic membrane. Among immunity-related transcripts, while lysozymes and lectins were the most highly expressed, several genes belonging to the Toll pathway - found at low levels in the gut of most insects - were identified, contrasting with a low abundance of transcripts from IMD and STAT pathways. Analysis of transcripts related to lipid metabolism indicates that lipids play multiple roles, being a major energy source, a substrate for perimicrovillar membrane formation, and a source for hydrocarbons possibly to produce the wax layer of the hindgut. Transcripts related to amino acid metabolism showed an unanticipated priority for degradation of tyrosine, phenylalanine, and tryptophan. Analysis of transcripts related to signaling pathways suggested a role for MAP kinases, GTPases, and LKBP1/AMP kinases related to control of cell shape and polarity, possibly in connection with regulation of cell survival, response of pathogens and nutrients. Together, our findings present a new view of the triatomine digestive apparatus and will help us understand trypanosome interaction and allow insights into hemipteran metabolic adaptations to a blood-based diet.
[Show abstract][Hide abstract] ABSTRACT: 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. The remarkable conservation of their predicted mature N-terminal region, which is exposed to the extracellular milieu, suggests that TcSMUG L products may be involved in structural and/or functional aspects of the interaction with the insect vector.
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. Our results indicate that the exogenous addition of TcSMUG L N-terminal peptide, but not control T. cruzi mucin peptides, to the infected bloodmeal inhibited the development of parasites in R. prolixus in a dose-dependent manner. Pre-incubation of insect midguts with the TcSMUG L peptide impaired the ex vivo attachment of epimastigotes to the luminal surface epithelium, likely by competing out TcSMUG L binding sites on the luminal surface of the posterior midgut, as revealed by fluorescence microscopy.
Together, these observations indicate that TcSMUG L mucins are a determinant of both adhesion of T. cruzi epimastigotes to the posterior midgut epithelial cells of the triatomine, and the infection of the insect vector, R. prolixus.
[Show abstract][Hide abstract] ABSTRACT: The present work is the second one of a two- part publication on the use
of Synchrotron Radiation Phase Contrast microtomography (SR-PhC-μCT)
for the study of Rhodnius prolixus morphology. In previous paper the
visualization of various detailed features were highlighted thanks to
the edge enhancement effects typical of the phase contrast technique,
but the contrast between foreground and background remained poor. In
this study the same data set have been re-visited with application of a
single distance phase retrieval algorithm. The resulting slices showed
very high quality images that enable a better visualization of important
muscles and neurohemal organs of the central nervous system within the
head of Rhodnius prolixus.
Journal of Instrumentation 07/2013; · 1.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have used phase-contrast X-ray microtomography (PPC-mCT) to study the head of the blood-feeding
bug, Rhodniusprolixus, whichisoneofthemostimportantinsectvectorof Trypanosomacruzi, ethiologic
processedby ANKA phase are comparedtothoseobtainedthroughdirecttomographicreconstructionof
[Show abstract][Hide abstract] ABSTRACT: Chagas disease is a trypanosomiasis whose causative agent is the protozoan parasite Trypanosoma cruzi, which is transmitted to humans by hematophagous insects known as triatomines and affects a large proportion of South America. The digestive tract of the insect vectors in which T. cruzi develops constitutes a dynamic environment that affects the development of the parasite. Thus, we set out to investigate the chemical composition of the triatomine intestinal tract through a metabolomics approach. We performed Direct Infusion Fourier Transform Ion Cyclotron Resonance Mass Spectrometry on fecal samples of three triatomine species (Rhodnius prolixus, Triatoma infestans, Panstrongylus megistus) fed with rabbit blood. We then identified groups of metabolites whose frequencies were either uniform in all species or enriched in each of them. By querying the Human Metabolome Database, we obtained putative identities of the metabolites of interest. We found that a core group of metabolites with uniform frequencies in all species represented approximately 80% of the molecules detected, whereas the other 20% varied among triatomine species. The uniform core was composed of metabolites of various categories, including fatty acids, steroids, glycerolipids, nucleotides, sugars, and others. Nevertheless, the metabolic fingerprint of triatomine feces differs depending on the species considered. The variable core was mainly composed of prenol lipids, amino acids, glycerolipids, steroids, phenols, fatty acids and derivatives, benzoic acid and derivatives, flavonoids, glycerophospholipids, benzopyrans, and quinolines. Triatomine feces constitute a rich and varied chemical medium whose constituents are likely to affect T. cruzi development and infectivity. The complexity of the fecal metabolome of triatomines suggests that it may affect triatomine vector competence for specific T. cruzi strains. Knowledge of the chemical environment of T. cruzi in its invertebrate host is likely to generate new ways to understand the factors influencing parasite proliferation as well as methods to control Chagas disease.
PLoS ONE 01/2013; 8(10):e77283. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We adapted the protocols of reducing sugar measurements with dinitrosalicylic and bicinchoninic acid for thermocyclers, and their use in enzymatic assays for hydrolases such as amylase and β-1,3-glucanase. The use of thermocyclers for these enzymatic assays resulted in a 10 times reduction in the amount of reagent and volume of the sample needed, when compared to conventional microplate protocols. We standardized absorbance readings from the PCR plates which allowed us to make direct readings of the techniques above, and a β-glycosidase assay was also established under the same conditions. Standardization of the enzymatic reaction in thermocyclers resulted in less time-consuming temperature calibrations, and without loss of volume through leakage or evaporation from the microplate. Kinetic parameters were successfully obtained and the use of the thermocycler allowed the measurement of enzymatic activities in biological samples from the field with a limited amount of protein.
[Show abstract][Hide abstract] ABSTRACT: Physalin B is a natural secosteroidal, extracted from the Solanaceae plant, Physalis angulata, and it presents immune-modulator effects on the bloodsucking bug, Rhodnius prolixus. In this work, R. prolixus was treated with physalin B at a concentration of 1mg/ml of blood meal (oral application), or 20ng/insect (applied topically) or 57ng/cm(2) of filter paper (contact treatment), and infected with Trypanosoma cruzi Dm28c clone (2×10(6) epimastigotes/insect). The three types of applications significantly decreased the number of T. cruzi Dm28c in the gut comparing with the non-treated infected insects (controls). All groups of infected insects treated with physalin B had higher numbers of bacterial microbiota in the gut than the non-treated controls infected with T. cruzi. We observed that the infected physalin B insects with topical and contact treatments had a lower antibacterial activity in the gut when compared with control infected insects. Furthermore, infected insects with the physalin B oral treatment produced higher levels of nitrite and nitrate in the gut than control infected insects. These results demonstrate that physalin B decreases the T. cruzi transmission by inhibiting the parasite development in the insect vector R. prolixus. Herein the importance of physalin B modulation on the immune system and microbiota population in terms of parasite development and transmission are discussed.
Journal of insect physiology 10/2012; · 2.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Trypanosoma rangeli is a protozoan that is non-pathogenic for humans and other mammals but causes pathology in the genus Rhodnius. T. rangeli and R. prolixus is an excellent model for studying the parasite-vector interaction, but its cycle in invertebrates remains unclear. The vector becomes infected on ingesting blood containing parasites, which subsequently develop in the gut, hemolymph and salivary glands producing short and large epimastigotes and metacyclic trypomastigotes, which are the infective forms. The importance of the T. rangeli cycle is the flagellate penetration into the gut cells and invasion of the salivary glands. The establishment of the parasite depends on the alteration of some vector defense mechanisms. Herein, we present our understanding of T. rangeli infection on the vector physiology, including gut and salivary gland invasions, hemolymph reactions and behavior alteration.
[Show abstract][Hide abstract] ABSTRACT: The effects of azadirachtin and ecdysone on the Trypanosoma cruzi population in the Rhodnius prolixus gut were investigated. T. cruzi were rarely found in the gut compartments of azadirachtin-treated larvae. High parasite numbers were observed in the stomach of the control and ecdysone groups until 10 days after treatment and in the small intestine and rectum until 25 days after treatment. High percentages of round forms developed in the stomachs of all groups, whereas azadirachtin blocked the development of protozoan intermediate forms. This effect was counteracted by ecdysone therapy. In the small intestine and rectum, epimastigotes predominated for all groups, but more of their intermediates developed in the control and ecdysone groups. Azadirachtin supported the development of round forms and their intermediates into trypomastigotes. In the rectum, trypomastigotes did not develop in the azadirachtin group and developed much later after ecdysone therapy. The parallel between the effects of azadirachtin and ecdysone on the host and parasite development is discussed on the basis of the present results because ecdysone appears to act directly or indirectly in determining the synchronic development of T. cruzi forms from round to epimastigotes, but not metacyclic trypomastigotes, in the invertebrate vector.
[Show abstract][Hide abstract] ABSTRACT: Trypanosoma cruzi in order to complete its development in the digestive tract of Rhodnius prolixus needs to overcome the immune reactions and microbiota trypanolytic activity of the gut. We demonstrate that in R. prolixus following infection with epimastigotes of Trypanosoma cruzi clone Dm28c and, in comparison with uninfected control insects, the midgut contained (i) fewer bacteria, (ii) higher parasite numbers, and (iii) reduced nitrite and nitrate production and increased phenoloxidase and antibacterial activities. In addition, in insects pre-treated with antibiotic and then infected with Dm28c, there were also reduced bacteria numbers and a higher parasite load compared with insects solely infected with parasites. Furthermore, and in contrast to insects infected with Dm28c, infection with T. cruzi Y strain resulted in a slight decreased numbers of gut bacteria but not sufficient to mediate a successful parasite infection. We conclude that infection of R. prolixus with the T. cruzi Dm28c clone modifies the host gut immune responses to decrease the microbiota population and these changes are crucial for the parasite development in the insect gut.
PLoS ONE 05/2012; 7(5):e36591. · 3.53 Impact Factor
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[Show abstract][Hide abstract] ABSTRACT: Chagas disease is one of the most important endemic diseases of South and Central America. Its causative agent is the protozoan Trypanosoma cruzi, which is transmitted to humans by blood-feeding insects known as triatomine bugs. These vectors mainly belong to Rhodnius, Triatoma and Panstrongylus genera of Reduviidae. The bacterial communities in the guts of these vectors may have important effects on the biology of T. cruzi. For this reason, we analyzed the bacterial diversity hosted in the gut of different species of triatomines using cultivation-independent methods. Among Rhodnius sp., we observed similar bacterial communities from specimens obtained from insectaries or sylvatic conditions. Endosymbionts of the Arsenophonus genus were preferentially associated with insects of the Panstrongylus and Triatoma genera, whereas the bacterial genus Serratia and Candidatus Rohrkolberia were typical of Rhodnius and Dipetalogaster, respectively. The diversity of the microbiota tended to be the largest in the Triatoma genus, with species of both Arsenophonus and Serratia being detected in T. infestans.
[Show abstract][Hide abstract] ABSTRACT: In recent years, the effects of pollution on the health of humans and other vertebrates were extensively studied. However, the effects on some invertebrates are comparatively unknown. Recent studies have demonstrated that toxic metals interfere with the reproduction, development and immune defenses of some terrestrial and marine invertebrates. Some environmental conditions including pollution produce chronic and acute effects on different animal's organs and systems. In this work, we investigated changes in the concentrations of Cl, K, Ca, Fe and Zn in Rhodnius prolixus as insect model. The elements were quantified using urine and hemolymph samples collected on different days after feeding the insects with blood containing HgCl2. The synchrotron radiation total reflection X-ray fluorescence measurements were carried at the X-ray fluorescence beamline facility in Brazilian Synchrotron Light Laboratory. The observation reveals that the calcium level was higher in the hemolymph than in urine. On the other hand, the urine collected from insects treated with HgCl2 showed higher level of Cl than hemolymph samples. Ca, Fe and Zn concentrations decrease drastically in urine samples collected after 2 days of HgCl2 treatment. The regulation of triatomines excretion was discussed pointing out the importance of trace elements.
Spectrochimica Acta Part B Atomic Spectroscopy 05/2012; s 71–72:127–130. · 3.15 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chagas disease is a trypanosomiasis whose agent is the protozoan parasite Trypanosoma cruzi, which is transmitted to humans by hematophagous bugs known as triatomines. Even though insecticide treatments allow effective control of these bugs in most Latin American countries where Chagas disease is endemic, the disease still affects a large proportion of the population of South America. The features of the disease in humans have been extensively studied, and the genome of the parasite has been sequenced, but no effective drug is yet available to treat Chagas disease. The digestive tract of the insect vectors in which T. cruzi develops has been much less well investigated than blood from its human hosts and constitutes a dynamic environment with very different conditions. Thus, we investigated the composition of the predominant bacterial species of the microbiota in insect vectors from Rhodnius, Triatoma, Panstrongylus and Dipetalogaster genera.
Microbiota of triatomine guts were investigated using cultivation-independent methods, i.e., phylogenetic analysis of 16s rDNA using denaturing gradient gel electrophoresis (DGGE) and cloned-based sequencing. The Chao index showed that the diversity of bacterial species in triatomine guts is low, comprising fewer than 20 predominant species, and that these species vary between insect species. The analyses showed that Serratia predominates in Rhodnius, Arsenophonus predominates in Triatoma and Panstrongylus, while Candidatus Rohrkolberia predominates in Dipetalogaster.
The microbiota of triatomine guts represents one of the factors that may interfere with T. cruzi transmission and virulence in humans. The knowledge of its composition according to insect species is important for designing measures of biological control for T. cruzi. We found that the predominant species of the bacterial microbiota in triatomines form a group of low complexity whose structure differs according to the vector genus.
[Show abstract][Hide abstract] ABSTRACT: c1 Corresponding author: Departamento de Biologia Geral, Instituto de Biologia, Universidade Federal Fluminense, Morro do Valonguinho s/n° Centro –Niterói, RJ, 24001-970, Brazil. Tel: +55 2126292285. Fax: +55 2126292376. E-mail: email@example.com