Near infrared spectroscopy accurately detects Trypanosoma cruzi non-destructively in midguts, rectum and excreta samples of Triatoma infestans.

Chagas disease is a neglected tropical disease caused by Trypanosoma cruzi parasite with an estimated 70 million people at risk. Traditionally, parasite presence in triatomine vectors is detected through optical microscopy which can be low in sensitivity or molecular techniques which can be costly in endemic countries. The aim of this study was to evaluate the ability of a reagent-free technique, the Near Infrared Spectroscopy (NIRS) for rapid and non-invasive detection of T. cruzi in Triatoma infestans body parts and in wet/dry excreta samples of the insect. NIRS was 100% accurate for predicting the presence of T. cruzi infection Dm28c strain (TcI) in either the midgut or the rectum and models developed from either body part could predict infection in the other part. Models developed to predict infection in excreta samples were 100% accurate for predicting infection in both wet and dry samples. However, models developed using dry excreta could not predict infection in wet samples and vice versa. This is the first study to report on the potential application of NIRS for rapid and non-invasive detection of T. cruzi infection in T. infestans in the laboratory. Future work should demonstrate the capacity of NIRS to detect T. cruzi in triatomines originating from the field.

Anti-Leishmania braziliensis activity of 1,10-phenanthroline-5,6-dione and its Cu(II) and Ag(I) complexes.

Leishmaniasis, included in the priority list of the WHO, remains as a neglected disease caused by parasites of the Leishmania genus. There is no vaccine available for human leishmaniasis, and the current treatment is based on old drugs that cause serious side effects. Herein, we initially studied the cellular distribution of the virulence factor gp63, the major metallopeptidase, in a virulent strain of Leishmania braziliensis, and then we measured the inhibitory effects of 1,10-phenanthroline-5,6-dione (phendione), and its metal complexes, [Cu(phendione)3](ClO4)2.4H2O and [Ag(phendione)2]ClO4, on both cellular and extracellular metallopeptidases produced by promastigotes. The action of the three compounds on parasite viability and on parasite-macrophage interaction was also determined. Gp63 molecules were detected in several parasite compartments, including the cytoplasm, the membrane lining the cell body and flagellum, and in the flagellar pocket, which explains the presence of gp63 in the culture medium. The test compounds inhibited parasite metallopeptidases in a typical dose-dependent manner, and they also caused a significant and irreversible inhibition of parasite motility. Moreover, the pre-treatment of promastigotes with the test compounds induced a decrease in the association index with macrophages. Collectively, phendione and its Cu(II) and Ag(I) complexes are excellent prototypes for the development of new anti-L. braziliensis drugs.

Morphology of the Eggs of Ornithocoris pallidus (Hemiptera, Cimicidae, Haematosiphoninae).

Morphology of cimicid eggs are scarce, and this is the first record for the genus Ornithocoris Pinto, 1927 (Hemiptera: Cimicidae), using scanning electron microscopy (SEM). This genus comprises two species: Ornithocoris toledoi Pinto, 1927 (Hemiptera: Cimicidae) and Ornithocoris pallidus (Usinger, 1959). The eggs of O. pallidus are ellipsoid, and an evident 'lateral flattening', which may give clear asymmetry from the longitudinal axis. The exochorion of the body of the egg and operculum of O. pallidus present spherical or polygonal structures in relief, and pseudomicropyles on the border of the operculum, both differing from Cimex lectularius. The internal face of the operculum is smooth and the border also presented three layers. It was not possible to observe micropyles in the egg of O. pallidus. Bed bugs have a great psychological impact on people, and some people can develop a more or less severe allergic reaction against the bite. Successful control of bed bugs needs serious organization. Studies on egg morphology will add more information to assist in taxonomy studies of O. pallidus and can serve as a basis for control studies because eggs are resistant to insecticides.

Morphological and ultrastructural analysis of an important place of sexual communication of Rhodnius prolixus (Heteroptera: Reduviidae): the Metasternal Glands.

Rhodnius prolixus is an important vector of Trypanosoma cruzi, the etiological agent of Chagas disease. Insect adults have a pair of Metasternal Glands (MGs) and the secretion emitted by these glands acts as sex pheromone. Recent studies have focused on the chemical composition of this pheromone, electrophysiological responses to MGs compounds and mating behavior assays. Morphological studies of these glands are still scarce. Thus, considering the relevance of MGs in the sex pheromone biosynthesis, we investigated the morphology and ultrastructure of R. prolixus MGs. The glandular apparatus presents a tubular structure containing secretory cells with canalicules that fuse with the central duct which conducts the secretion to a pear-shaped reservoir connected to the exterior by a droplet-shape orifice. The secretory cells are classified as class III, they present a well-developed rough and smooth endoplasmic reticulum. Smooth endoplasmic reticulum is a site of lipid biosynthesis that may be involved in the mevalonate pathway, a probable route of the sex pheromone biosynthesis in this insect. The presence of rough endoplasmic reticulum indicates a possible peptides/proteins secretions site which were still not characterized in MGs. Several mitochondria are scattered in the cytoplasm that may suggest a high metabolic activity. Further studies should be carried out to correlate these data with the sex pheromone biosynthesis in this vector.

Development of conventional and real-time multiplex PCR-based assays for estimation of natural infection rates and Trypanosoma cruzi load in triatomine vectors.

BACKGROUND: Chagas disease is a complex anthropozoonosis with distinct domestic and sylvatic mammal species acting as potential reservoirs. The diversity of vector species and their habitats are among the factors that hinder the control of the disease. Control programs periodically monitor the prevalence of T. cruzi infection in insect bugs through microscopical observation of diluted feces. However, microscopy presents limited sensitivity in samples with low parasite numbers, difficulties in examining all evolutionary stages of the insect and may in turn be limited to differentiate T. cruzi from other morphologically similar trypanosomatids. Here, we report two highly sensitive and accurate methodologies to infer T. cruzi infection rates and to quantify parasite load in the gut of field-collected triatomines. METHODS: Triatomines were manually collected in the period 2011-2012 and 2014-2015, in domestic, peridomestic or sylvatic habitats in rural areas of 26 municipalities, encompassing three distinct Brazilian biomes: Caatinga, Cerrado and Atlantic Rainforest. Following morphological and taxonomical identification, the search for flagellated protozoa was performed by optical microscopy. A conventional PCR targeting T. cruzi kDNA and a TaqMan qPCR directed to the parasite nuclear satellite DNA (SAT) were developed, both in multiplex, with the triatomine 12S subunit ribosomal RNA gene, used as internal amplification control. Both methods were used for detection (kDNA-PCR) and parasite load quantification (SAT-DNA-qPCR), to investigate T. cruzi infection in captured triatomines. RESULTS: The combined methods were assayed on a panel of 205 field-collected triatomine samples. Diagnostic analysis revealed 21% positivity for the kDNA-PCR, whereas microscopic examination enabled identification of T. cruzi in only 7.0% of the PCR-positive samples. Negative PCR results were confirmed by the absence of T. cruzi flagellates using microscopy. Caatinga biome yielded the highest T. cruzi infection rate (60%), followed by the Atlantic Rainforest and Cerrado with 7.1 and 6.1%, respectively. In addition, a wide range distribution of parasite load, varying from 8.05 × 10-2 to 6.31 × 1010 was observed with a median of 2.29 × 103 T. cruzi/intestine units. When parasite load was analyzed by triatomine species, a significantly higher median was found for Panstrongylus lutzi in comparison with Triatoma brasiliensis. CONCLUSIONS: Our results demonstrate highly sensitive PCR-based methodologies to monitor T. cruzi infection in triatomines. In addition, the qPCR assay offers the possibility of further evaluation parasite load, as a promising biomarker of the vectorial capacity of triatomines in Chagas disease endemic areas.

Larvicidal activity and effects on post embrionary development of laboratory reared Musca domestica (Linnaeus, 1758) (Diptera: Muscidae), treated with Brevibacillus laterosporus (Laubach) spore suspensions.

The application of a spore suspension of Brevibacillus laterosporus (Laubach) (strain Bon707), at a concentration of 1.94×10(9)CFU/mL in the diet, induced a level of 70% mortality in larvae of Musca domestica. No sublethal effects, upon feeding activity or development were recorded. However, electron microscopic examination of the digestive tract of larvae fed with B. laterosporus, revealed cellular vacuolization and cytoplasmic disorganization.

Evaluation of larvicidal activity and effects on post embrionary development of laboratory reared Lucilia cuprina (Wiedemann, 1830) (Diptera: Calliphoridae), treated with Brevibacillus laterosporus.

The blowfly Lucilia cuprina is an economically important livestock pest that is also associated with human myiasis. To date, methods including the application of chemical pesticides, plant extracts, insect growth regulators and a range of Bacillus thuringiensis strains have been used, with varying degrees of success, to control this pest. The present study evaluated the larvicidal activity and the induction of sub lethal effects upon post embrionary development following ingestion of 12 strains of Brevibacillus laterosporus, presented individually in the diet as spores. All strains were shown to be larvicidal, with corrected mortality levels of 29 to 54%. No significant differences were observed, in terms of larval weight at the time of abandoning the diet, in the time taken for the initiation of the pupation process, in the duration of the pupation process, the period of adult emergence or cumulative mortality during the transition from larvae to adult. However, an influence upon sex ratio was observed. This study suggests that strains of B. laterosporus hold potential for development as a tool in the biological control of L. cuprina.

Culicidae community composition and temporal dynamics in Guapiaçu Ecological Reserve, Cachoeiras de Macacu, Rio de Janeiro, Brazil.

A temporal observational study was conducted of the Culicidae fauna in a remnant area of Atlantic Forest within a private reserve (Guapiaçu Ecological Reserve-REGUA) presenting typical vegetation cover of dense rain forest, with some patches recovering a floristic composition similar to that of the original community. Research was carried out to analyze the influence of climatic factors (mean monthly temperature, rainfall, and air relative humidity) on the temporal dynamics of the mosquito communities that occur in the reserve. The completeness of the mosquito inventory was assessed with individual-based rarefaction-extrapolation curves. Differences in species composition between sites and months were tested with PERMANOVA. True diversities of orders 0, 1, and 2 (effective numbers) were estimated and compared between sites, months, and years. Multiple stepwise regressions were used to assess relationships between climatic variables, measures of diversity, and abundances of the most common species. There were significant interactive effects between year and site on measures of diversity. However, diversity estimates showed little variation among months, and these were weakly correlated with climatic variables. Abundances of the most common species were significantly related to temperature or relative humidity, but not rainfall. The presence of mosquito species known to be vectors of human diseases combined with an intermittent flow of visitors to the study area suggests there is a risk of disease transmission that warrants further monitoring.

Anatomy of the spermatophore in triatomines (Hemiptera, Reduviidae, Triatominae) and its applications to the study of Chagas disease vector biology.

The present study focused on spermatophore structure, transfer, and subsequent destination inside bloodfeeding females of the species Triatoma infestans and Rhodnius neglectus. The morphology of the spermatophore differed between the species studied, such that in T. infestans, the shape was ovaloid, whereas in R. neglectus, the shape resembled a rod. Structures' spine-like cuticulars distributed across the inner surface of the vagina of both species were observed; however, the role of these cuticulars is unknown in Triatominae. In both species, there was an opening in the spermatophore exactly where the common oviduct is connected, thereby making it possible to confirm that the process of spermatozoid migration takes place through this opening. The results obtained show that the spermatophores of T. infestans and R. neglectus differ in size, shape, and structure. Therefore, they can be used as taxonomic markers and may provide information regarding physiology and evolution.

Correlation between populations of Rhodnius and presence of palm trees as risk factors for the emergence of Chagas disease in Amazon region, Brazil.

Among the states that comprise the legally defined Amazon region of Brazil, Pará has presented the highest occurrences of acute cases of Chagas disease over the last two decades. These cases have been attributed to consumption of fruits from native palm trees. In surveys in rural and wild areas of the municipality of Oriximiná, Pará, triatomine fauna, their main ecotopes and the infection rate due to Trypanosoma cruzi were identified using active and passive search methods: manual capture and Noireau traps, respectively. A total of 582 ecotopes were surveyed using 1496 Noireau traps. Out of 442 specimens collected, 289 were identified as Rhodnius robustus and 153 as Rhodnius pictipes. The infection rate caused by T. cruzi was 17.4%. The food sources of the triatomines were found to be birds, hemolymph, horses, and rodents. The association between R. robustus and inajá palm trees (Attalea marita), which are abundant in rural areas, was confirmed. On the other hand, R. pictipes is found in several palm tree species, such as inajá (A. marita), mucajá (Acrocomia aculeata), murumuru (Astrocaryum murumuru) and patauá (Oenocarpus bataua), and in bromeliads in wild areas. These occurrences of triatomine species in regions with or without T. cruzi infection, in the vicinity of the main settlement of the municipality, suggest that there is a need for entomological and epidemiological surveillance in this region.

Grandisin caused morphological changes larval and toxicity on Aedes aegypti

Dengue is a tropical disease caused by an arbovirus transmitted by Aedes aegypti. Since no effective vaccine is available for treating dengue, the present study focused on population vector control through investigating the use of the lignan grandisin, isolated from Piper solmsianum C. DC., Piperaceae, against the larvae of A. aegypti. Grandisin caused larval (L3) mortality at LC50 150 µg/mL. Histological analysis on A. aegypti larvae treated with grandisin (LC50 50 µg/mL) showed changes in the anterior-middle midgut, with intense tissue destruction and cell disorganization.

Ultrastructural localization of basic proteins and carbohydrates in male accessory glands of two Triatoma species (Hemiptera, Reduviidae, Triatominae).

The male accessory glands in Triatoma are tubular and produce substances with some functions related to production of the spermatophore. In the current study, the cytochemistry of male accessory glands was evaluated in starved Triatoma brasiliensis and adult Triatoma melanica. The storage of carbohydrates and proteins in T. melanica male accessory glands occurs earlier than in T. brasiliensis. In addition, the occurrence of eletron-lucent granules without carbohydrates and proteins suggests that other substances are released by these glands, which may be used for lubrication of the male genitalia. Male T. brasiliensis has more intense secretory activity in the fifth day of adult life, which may indicate a higher reproductive capacity. The analysis of lipid production in male accessory glands can contribute to the knowledge of spermatophore formation in these species.

CdTe and CdSe quantum dots cytotoxicity: a comparative study on microorganisms.

Quantum dots (QDs) are colloidal semiconductor nanocrystals of a few nanometers in diameter, being their size and shape controlled during the synthesis. They are synthesized from atoms of group II-VI or III-V of the periodic table, such as cadmium telluride (CdTe) or cadmium selenium (CdSe) forming nanoparticles with fluorescent characteristics superior to current fluorophores. The excellent optical characteristics of quantum dots make them applied widely in the field of life sciences. Cellular uptake of QDs, location and translocation as well as any biological consequence, such as cytotoxicity, stimulated a lot of scientific research in this area. Several studies pointed to the cytotoxic effect against micoorganisms. In this mini-review, we overviewed the synthesis and optical properties of QDs, and its advantages and bioapplications in the studies about microorganisms such as protozoa, bacteria, fungi and virus.

Influence of leishmanolysin-like molecules of Herpetomonas samuelpessoai on the interaction with macrophages.

Monoxenous trypanosomatids usually have an invertebrate as the only host in their life cycles, however, they have been found repeatedly in plants and/or mammals. To succeed in colonizing a vertebrate host, the parasite must quickly adapt to drastic changes in the environment (e.g. temperature), which reflect the conditions found in the insect and mammalian hosts. Leishmanolysin is a metalloprotease ubiquitously distributed in trypanosomatids, playing a myriad of functions. In Herpetomonas samuelpessoai, an insect trypanosomatid, the leishmanolysin-like molecule was implicated in the nutrition and insect adhesion. Herein, we showed that leishmanolysin expression is equally expressed in H. samuelpessoai parasites submitted to insect (26 °C) and mammalian (37 °C) temperatures. Also, the parasites grown in both temperatures interacted at similar rates with macrophages. Finally, we showed that leishmanolysin is involved in crucial steps in the interaction of H. samuelpessoai cells with macrophages, since the treatment with either anti-leishmanolysin antibodies or metalloprotease inhibitor 1,10-phenanthroline significantly reduced the association index. Similarly, the treatment of the macrophages with purified leishmanolysin promoted a powerful reduction in the association index, suggesting the direct involvement of macrophage receptors. These results suggest that H. samuelpessoai leishmanolysin molecules are not modulated by temperature and are involved in the interaction with mammalian cells.

Leishmanolysin-like molecules in Herpetomonas samuelpessoai mediate hydrolysis of protein substrates and interaction with insect.

Herpetomonas samuelpessoai, an insect trypanosomatid, produces a 63-kDa metallopeptidase that has similar biochemical/immunological properties to Leishmania leishmanolysin, a virulence factor that participates in different stages of the parasite life cycle. Herein, we described some biochemical characteristics of the major surface metallopeptidase of H. samuelpessoai that led us to infer some probable functions for this peptidase during the parasite-invertebrate interaction. Gelatin-SDS-PAGE, flow cytometry and confocal fluorescence microscopy provided measurements for the relative levels of surface leishmanolysin-like molecules in H. samuelpessoai. Immunocytochemical analysis demonstrated the presence of leishmanolysin-like molecules on the surface and cytoplasm of the parasite. The surface metallopeptidase was active at a broad spectrum of pH and temperature, showing maximum activity at pH 6.0 at 37 degrees C, and an ability to degrade albumin, hemoglobin, IgG, mucin, casein and gut proteins obtained from Aedes aegypti. This wide substrate utilization might support parasite growth and development. Curiously, H. samuelpessoai cells were able to colonize A. aegypti guts. In an effort to implicate a possible role for the metallopeptidase from H. samuelpessoai, living parasites were treated with different compounds before the interaction with gut cells. The pre-incubation with metallopeptidase inhibitors, phospholipase C or anti-leishmanolysin antibodies promoted a significant reduction in the interaction with guts. Similarly, the pre-treatment of gut cells with purified leishmanolysin-like protein drastically diminished the adhesion process. Furthermore, the expression of surface leishmanolysin in H. samuelpessoai cells was drastically enhanced after passage in A. aegypti. These results suggest the participation of homologues of leishmanolysin in the interaction of H. samuelpessoai with the invertebrate vector.

In vitro and in vivo documentation of quantum dots labeled Trypanosoma cruzi--Rhodnius prolixus interaction using confocal microscopy.

Semiconductor quantum dots (QDs) are highly fluorescent nanocrystals markers that allow long photobleaching and do not destroy the parasites. In this paper, we used fluorescent core shell quantum dots to perform studies of live parasite-vector interaction processes without any observable effect on the vitality of parasites. These nanocrystals were synthesized in aqueous medium and physiological pH, which is very important for monitoring live cells activities, and conjugated with molecules such as lectins to label specific carbohydrates involved on the parasite-vector interaction. These QDs were successfully used for the study of in vitro and in vivo interaction of Trypanosoma cruzi and the triatomine Rhodnius prolixus. These QDs allowed us to acquire real time confocal images sequences of live T. cruzi-R. prolixus interactions for an extended period, causing no damage to the cells. By zooming to the region of interest, we have been able to acquire confocal images at the three to four frames per second rate. Our results show that QDs are physiological fluorescent markers capable to label living parasites and insect vector cells. QDs can be functionalized with lectins to specifically mark surface carbohydrates on perimicrovillar membrane of R. prolixus to follow, visualize, and understand interaction between vectors and its parasites in real-time.

Molecular characterization of Trypanosoma cruzi sylvatic isolates from Rio de Janeiro, Brazil.

Trypanosoma cruzi, the etiologic agent of Chagas disease, presents considerable heterogeneity between isolated populations within the wild and domestic cycles. By using multiplex polymerase chain reaction based on the mini-exon gene, characterization was performed on seven samples isolated from specimens of Triatoma vitticeps that had been collected from the locality of Triunfo in the municipality of Santa Maria Madalena, state of Rio de Janeiro, Brazil. The samples SMM10, SMM53, SMM88, and SMM98 (area A) and SMM36 and SMM82 (area B) revealed the presence of 150 base pairs, corresponding to the zymodeme III (Z3). Our study suggested that one isolate (SMM1) presents a mixed genotype associated with Z3 and TcII. The typing of isolates of T. cruzi has the main aim of identifying strains with different epidemiological and/or clinical characteristics of Chagas disease. Our results corroborate other descriptions in the literature and contribute towards the knowledge and records of the profile of some additional wild isolates of T. cruzi in regions not yet affected by the disease.

Trypanosoma rangeli: Differential expression of cell surface polypeptides and ecto-phosphatase activity in short and long epimastigote forms.

Trypanosoma rangeli is a parasite of a numerous wild and domestic animals, presenting wide geographical distribution and high immunological cross-reactivity with Trypanosoma cruzi, which may lead to misdiagnosis. T. rangeli has a complex life cycle, involving distinct morphological and functional forms in the vector. Here, we characterized the cell surface polypeptides and the phosphatase activities in short and long epimastigotes forms of T. rangeli, using intact living parasites. The surface protein profile revealed by the incubation of parasites with biotin showed a preferential expression of the 97, 70, 50, 45, 25-22, and 15 kDa biotinylated polypeptides in the long forms, in contrast to the 55 and 28 kDa biotinylated polypeptides synthesized by the short epimastigotes. Additionally, flow cytometry analysis showed that the short forms had relatively lower biotin surface binding than long ones. The involvement of phosphatases with the trypanosomatid differentiation has been proposed. In this sense, T. rangeli living parasites were able to hydrolyze the artificial substrate p-nitrophenylphosphate at a rate of 25.57+/-2.03 and 10.09+/-0.93 nmol p-NPP x h(-1) x 10(7) cells for the short and long epimastigotes, respectively. These phosphatase activities were linear with time for at least 60 min and the optimum pH lies in the acid range. Classical inhibitors of acid phosphatases, such as ammonium molybdate, sodium fluoride, and zinc chloride, showed a significant decrease in these phosphatase activities, with different patterns of inhibition. Additionally, these phosphatase activities presented different kinetic parameters (Km and Vmax) and distinct sensitivities to divalent cations. Both epimastigotes were unable to release phosphatase to the extracellular environment. Cytochemical analysis demonstrated the localization of these enzymes on the parasite surfaces (cell body and flagellum) and in intracellular vacuoles, resembling acidocalcisomes.

Scanning electron microscopy study of the egg of Haemagogus (Haemagogus) capricornii Lutz, 1904 (Diptera: Culicidae).

Morphological details are provided for the dorsal and ventral surfaces of both extremities and the micropylar area of eggs of Haemagogus (Haemagogus) capricornii Lutz, captured in the Biological Reserve of Tinguá, State of Rio de Janeiro, Brazil. The eggs were observed by scanning electron microscopy with a morphometrical analysis of the main structures. The outer chorionic cells on the ventral surface were extremely regular, such as those observed in Hg. equinus and Hg. janthinomys. The tubercles present differences in form, size, and distribution. Filaments to attach to the substrate were observed in this species.

Scanning electron microscopy of eggs of Ochlerotatus (Protomacleaya) terrens Walker.

Observations on the morphological ultrastructure of eggs of Ochlerotatus (Protomacleaya) terrens (Diptera: Culicidae) were conducted by using scanning electron microscopy. Morphometry of the principal structures was obtained with the aid of Semafore analysis software. Eggs of Oc. terrens from females caught in the Biological Reserve of Tinguá, State of Rio de Janeiro, Brazil, were utilized. The eggs presented elliptical outlines, with a length of approximately 649.0 microm and width of 168.7 microm. The egg index (length to width ratio) was 3.85 microm. The exochorion had hexagonal and sometimes pentagonal ornamentation. Inside the chorionic cells were small, well-distributed tubercles with a large range of sizes. The micropylar apparatus, located in the anterior region of the egg, presented a collar with a poorly visible frame, with borders of indeterminate extent and margins without a defined transition area, and a thickness of approximately 1.8 microm. The micropyle was plugged.