Interaction between Trypanosoma rangeli and the Rhodnius prolixus salivary gland depends on the phosphotyrosine ecto-phosphatase activity of the parasite

Trypanosoma rangeli is the trypanosomatid that colonizes the salivary gland of its insect vector, with a profound impact on the feeding capacity of the insect. In this study we investigated the role of the phosphotyrosine (P-Tyr) ecto-phosphatase activity of T. rangeli in its interaction with Rhodnius prolixus salivary glands. Long but not short epimastigotes adhered to the gland cells and the strength of interaction correlated with the enzyme activity levels in different strains. Differential interference contrast microscopy demonstrated that clusters of parasites are formed in most cases, suggesting cooperative interaction in the adhesion process. The tightness of the correlation was evidenced by modulating the P-Tyr ecto-phosphatase activity with various concentrations of inhibitors. Sodium orthovanadate, ammonium molybdate and zinc chloride decreased the interaction between T. rangeli and R. prolixus salivary glands in parallel. Levamisole, an inhibitor of alkaline phosphatases, affected neither process. EDTA strongly inhibited adhesion and P-Tyr ecto-phosphatase activity to the same extent, an effect that was no longer seen if the parasites were pre-incubated with the chelator and then washed. When the P-Tyr ecto-phosphatase of living T. rangeli epimastigotes was irreversibly inactivated with sodium orthovanadate and the parasite cells were then injected into the insect thorax, colonization of the salivary glands was greatly depressed for several days after blood feeding. Addition of P-Tyr ecto-phosphatase substrates such as p-nitrophenyl phosphate (pNPP) and P-Tyr inhibited the adhesion of T. rangeli to salivary glands, but P-Ser, P-Thr and β-glycerophosphate were completely ineffective. Immunoassays using anti-P-Tyr-residues revealed a large number of P-Tyr-proteins in extracts of R. prolixus salivary glands, which could be potentially targeted by T. rangeli during adhesion. These results indicate that dephosphorylation of structural P-Tyr residues on the gland cell surfaces, mediated by a P-Tyr ecto-phosphatase of the parasite, is a key event in the interaction between T. rangeli and R. prolixus salivary glands.     

Suppression of the prophenoloxidase system in Rhodnius prolixus orally infected with Trypanosoma rangeli

Investigations were carried out to compare aspects of the prophenoloxidase (proPO)-activating pathway in Rhodnius prolixus hemolymph in response to oral infection and inoculation of the insects with two developmental forms of Trypanosoma rangeli epimastigotes strain H14. In vivo experiments demonstrated that in control insects fed on uninfected blood, inoculation challenge with short epimastigotes resulted in high phenoloxidase (PO) activity. In contrast, previous feeding on blood containing either short or long epimastigotes was able to suppress the proPO activation induced by thoracic inoculation of the short forms. In vitro assays in the presence of short epimastigotes demonstrated that control hemolymph or hemolymph provided by insects previously fed on blood containing epimastigotes incubated with fat body homogenates from control insects significantly increased the PO activity. However, fat body homogenates from insects previously fed on blood containing epimastigotes, incubated with hemolymph taken from insects fed on control blood or blood infected with epimastigotes, drastically reduced the proPO activation. The proteolytic activity in the fat body homogenates of control insects was significantly higher than in those obtained from fat body extracts of insects previously fed on blood containing epimastigotes. These findings indicate that the reduction of the proteolytic activities in the fat body from insects fed on infected blood no longer allows a significant response of the proPO system against parasite challenge. It also provides a better understanding of T. rangeli infection in the vector and offer novel insights into basic immune processes in their invertebrate hosts.     

The interaction between Trypanosoma rangeli and the nitrophorins in the salivary glands of the triatomine Rhodnius prolixus (Hemiptera; Reduviidae)

The parasite Trypanosoma rangeli develops in the intestinal tract of triatomines and, particularly in species of the genus Rhodnius, invades the hemolymph and salivary glands, where subsequent metacyclogenesis takes place. Many aspects of the interaction between T. rangeli and triatomines are still unclear, especially concerning the development of the parasite in the salivary glands and how the parasite interacts with the saliva. In this work, we describe new findings on the process of T. rangeli infection of the salivary glands and the impact of infection on the saliva composition. To ensure a complete infection (intestinal tract, hemolymph and salivary glands), 3rd instar Rhodnius prolixus nymphs were fed on blood containing T. rangeli epimastigotes using an artificial feeder. After molt to the 4th instar, the nymphs were inoculated with epimastigotes in the hemolymph. The results showed that the flagellates started to invade the salivary glands by the 7th day after the injection. The percentage of trypomastigotes inside the salivary glands continuously increased until the 25th day, at which time the trypomastigotes were more than 95% of the T. rangeli forms present. The salivary contents from T. rangeli-infected insects showed a pH that was significantly more acidic (<6.0) and had a lower total protein and hemeprotein contents compared with non-infected insects. However, the ratio of hemeprotein to total protein was similar in both control and infected insects. qPCR demonstrated that the expression levels of three housekeeping genes (18S rRNA, β-actin and α-tubulin) and nitrophorins 1–4 were not altered in the salivary glands after an infection with T. rangeli. In addition, the four major nitrophorins (NPs 1–4) were knocked down using RNAi and their suppression impacted T. rangeli survival in the salivary glands to the point that the parasite burden inside the R. prolixus salivary glands was reduced by more than 3-fold. These results indicated that these parasites most likely non-specifically incorporated the proteins that were present in R. prolixus saliva as nutrients, without impairing the biosynthesis of the antihemostatic molecules.     

Effects of gamma irradiation on the development of Trypanosoma rangeli in the vector Rhodnius prolixus

Studies on the effects of gamma radiation on the infectivity of Trypanosoma rangeli (strain H14) for the vector Rhodnius prolixus revealed that (i) the LD(50) (lethal dose for 50% of bugs) for uninfected insects was 4147 rads; (ii) irradiated insects with a dose of 1200 rads subsequently infected with the flagellates exhibited a mortality of 45%, while uninfected irradiated insects showed a mortality of 5%, and infected nonirradiated insects exhibited 10% mortality; (iii) flagellates were present in the hemolymph of irradiated insects 7 days postinfection (p.i.), while in nonirradiated insects the parasites appeared in the hemocoel 18 days p.i.; (iv) T. rangeli infection decreased the number of hemocytes significantly and induced the formation of nodules in the hemolymph of both irradiated and nonirradiated insects; and (v) gamma irradiation affected the ultrastructural organization of the epithelial cells of the small intestine, principally the perimicrovillar membranes and microvilli. In this paper, we discuss the significance of the intestinal microenvironment of R. prolixus with regard to its interaction with T. rangeli.     

Further morphological studies on the behavior of Trypanosoma rangeli in the hemocytes of Rhodnius prolixus

The hemocytes of Rhodnius prolixus were analyzed during the course of infection with the protozoan Trypanosoma rangeli. The following cell types were identified: prohemocyte, plasmatocyte, adipocyte, granular cell and oenocytoid. The number of these cells changes during the infection course thus indicating a cell response to infection of R. prolixus by T. rangeli. Transmission electron microscopy showed that plasmatocytes were able to ingest epimastigote forms of the parasite, which were then found within a parasitophorous vacuole. Amorphous material was seen within the vacuole suggesting that fusion of host cell lysosomes with the vacuole took place. Intravacuolar parasites in process of digestion were observed. In addition, reaction product indicative of the presence of acid phosphatase was observed in parasite-containing vacuoles. No dividing parasites were seen within the vacuole in contrast to what was observed outside the host cells.     

Rhodnius prolixus infected with Trypanosoma rangeli: In vivo and in vitro experiments

Studies were carried out on the activation of the prophenoloxidase (proPO) in adults of Rhodnius prolixus infected by short and long epimastigote forms of Trypanosoma rangeli. The in vitro activation of the proPO cascade using l-DOPA as substrate was very low in the absence of fat body extract, hemolymph, and parasites. On the other hand, a higher PO activity was observed when short, but not long, epimastigotes of T. rangeli were incubated with fresh hemolymph, fat body extract, and l-DOPA. Supernatant from lysed long epimastigotes increased the PO activity at levels identical to those observed with supernatants from lysed short epimastigotes. Similarly, the PO activity of hemolymph obtained from inoculated insects with long epimastigotes of T. rangeli showed a very low activity when incubated with l-DOPA compared to the PO activity of hemolymph taken from insects inoculated with short epimastigotes of T. rangeli. Control insects inoculated with sterile PBS showed no PO activity. These data indicate the presence of (a) factor(s) in the hemolymph as well as in the fat body extract that may be released (or induced) by the presence of short epimastigotes of T. rangeli and which results in the activation of the R. prolixus proPO system. The implications of these findings are discussed in relation to the development of T. rangeli and its ability to overcome the proPO system, survive, and successfully colonize the hemolymph of R. prolixus.     

Trypanosoma cruzi: involvement of glycoinositolphospholipids in the attachment to the luminal midgut surface of Rhodnius prolixus

Trypanosoma cruzi epimastigotes adhere in vivo to the luminal surface of their triatomid vector digestive tract by molecular mechanisms, as yet, unknown. Here, we show that the administration of 0.5 microM epimastigote major surface glycoinositolphospholipids (GIPLs) to the infected bloodmeal inhibits up to 90% parasite infection in Rhodnius prolixus. The parasite behavior was investigated in vitro using fragments of the insect midgut. The addition of GIPLs in concentration as low as 50-100 nM impaired 95% the attachment of epimastigotes. Previous treatment of GIPLs with trifluoroacetic acid to remove the terminal beta-galactofuranosyl residues reversed 50% the epimastigote in vitro attachment. The binding sites of purified GIPLs on the luminal surface of the posterior midgut were exposed by immunofluorescence microscopy. These observations indicate that GIPLs are one of the components involved in the adhesion of T. cruzi to the luminal insect midgut surface and possibly one of the determinants of parasite infection in the insect vector.     

An electron microscopic study of penetration by Trypanosoma rangeli into midgut cells of Rhodnius prolixus

The process of interaction of the Choachi strain of Trypanosoma rangeli with intestinal epithelial cells of Rhodnius prolixus was analyzed in experiments carried out in vitro and in vivo. For the in vitro experiments small fragments of the anterior region of the posterior midgut were incubated in the presence of the parasites, fixed, and processed for observation with the scanning electron microscope. Parasites attached to the surface of some epithelial cells, especially to the extracellular membrane layers (perimicrovillar membranes), were observed. For the in vivo experiments insects were infected with cultures of T. rangeli, sacrificed at different time intervals, and then processed for scanning and transmission electron microscopy. An intimate contact between the parasites and the membrane layers was observed. The parasites penetrated into cells that showed an electronlucent cytoplasm and a damaged surface, moved within the cytoplasm of the epithelial cell, reached the basal region, crossed the basal lamina, and entered the hemocoel.     

Inhibition of hemocyte microaggregation reactions in Rhodnius prolixus larvae orally infected with Trypanosoma rangeli

Hemocoelic inoculation of epimastigotes of Trypanosoma rangeli strain H14 into 5th-instar larvae of Rhodnius prolixus previously fed on blood containing the same parasites, showed reduced number of hemocyte microaggregates in the hemolymph, enhanced number of flagellates in the hemolymph as well as increased mortality of these insects. All these effects were counteracted by combined inoculation of R. prolixus with T. rangeli and arachidonic acid. In vitro assays using hemolymph taken from insects previously fed on blood containing parasites showed that hemocyte microaggregation reactions were also attenuated when T. rangeli is used as inducer of the reaction, and that simultaneous applying T. rangeli with arachidonic counteracted the hemocyte microaggregation inhibition. We suggest that arachidonic acid pathway can be a mediator of hemocyte microaggregation reactions in the hemolymph of insects inoculated with T. rangeli, and that oral infection with this protozoan inhibits the release of arachidonic acid.     

Studies on a haemolymph lectin isolated from Rhodnius prolixus and its interaction with Trypanosoma rangeli

We demonstrated that in Rhodnius prolixus haemocyte monolayers, both Trypanosoma cruzi and Trypanosoma rangeli are capable of inducing haemocyte/parasite clump formation. We also purified, by one-step affinity chromatography, a haemolymph galactoside-binding lectin from R. prolixus which we believe could play an important role in the development of T. rangeli in the haemocoel of the insect vector. This lectin markedly enhanced the activation of clump formation by T. rangeli in R. prolixus haemocyte monolayers, with an increase in clump size and haemocyte aggregation. The haemolymph lectin also significantly affected the motilitity and survival of T. rangeli culture short forms, but not the long forms, when they were incubated in vitro. This molecule is also one of the few described in insects with agglutination activity independent of calcium ions. The partial N-terminal amino acid sequence of this lectin demonstrated similarity to a bacterial xylulose kinase and in preliminary experiments the purified haemolymph lectin phosphorylated a tyrosine kinase substrate in a dose-dependent manner. The possible role of this haemolymph lectin in the life cycle of T. rangeli is discussed.     

Studies on Trypanosoma rangeli Tejera, 1920. VI. Developmental pattern in the haemolymph of Rhodnius prolixus

The morphological sequence of Trypanosoma rangeli development in the body cavity of Rhodnius prolixus is described. The metacyclic trypanosome is the product of successive division and transformation during the intra and extracellular development in the haemocoele. The significance of the early invasion of T. rangeli into the haemolymph is discussed. The epidemiological importance of the developmental pattern of T. rangeli in the vectors haemolymph and the host-response to the parasite are considered.     

Trypanosoma rangeli: effects of physalin B on the immune reactions of the infected larvae of Rhodnius prolixus

Physalins are seco-steroids obtained from plants of the family Solanaceae. Herein, we tested Physalis angulata L purified physalin B as an immunomodulatory compound in 5th-instar larvae of Rhodnius prolixus, which were systemically infected with the H14 Trypanosoma rangeli strain protozoan. In uninfected insects, the effective concentration of physalin B, which inhibited 50% of the blood ingested (ED(50)) volume, was 15.2+/-1.6 microg/ml of the meal. Ecdysis processes and mortality in uninfected larvae, treated orally with physalin B in concentrations ranging from 1 to 10 microg/ml, was similar to that observed in insects not treated with physalin B. However, R. prolixus larvae previously fed on blood containing 1.0, 0.1, and 0.01 microg of physalin B/ml exhibited mortality rates of 78.1, 54.3, and 12.7%, respectively, 6 days after inoculation of T. rangeli (1 x 10(3) parasites/insect), whereas only 7.2% mortality was observed in the control group, injected with sterile culture medium. The insects treated with physalin B (0.1 microg/ml) and inoculated with T. rangeli did not modify the phenoloxidase (PO) activity and total hemocyte count in the hemolymph. However, physalin B treatment caused a reduction in hemocyte micro-aggregation and nitric oxide production and enhanced the parasitemia in the hemolymph. These results demonstrate that physalin B from P. angulata is a potent immunomodulatory substance for the bloodsucking insect, R. prolixus.     

Role of superoxide and reactive nitrogen intermediates in Rhodnius prolixus (Reduviidae)/Trypanosoma rangeli interactions.

This study compares aspects of the superoxide, nitric oxide and prophenoloxidase pathways in Rhodnius prolixus hemolymph, measured in parallel, in response to Trypanosoma rangeli inoculation. Responses to two strains of T. rangeli, and two developmental forms, were studied, and the results obtained were correlated with the ability of the parasites to survive, multiply, and complete their life cycles in the hemolymph of the host. T. rangeli H14 strain parasites, which fail to complete their life cycle in Rhodnius by invading the salivary glands, stimulated high levels of superoxide and prophenoloxidase activity, which peaked 24 h after inoculation. Simultaneously, the concentration of hemolymph nitrites and nitrates increased, indicative of nitric oxide activity, but parasite numbers remained low. T. rangeli Choachi strain parasite inoculation also stimulated superoxide and prophenoloxidase activity, which, though significantly lower than the equivalent responses to the H14 strain, also peaked at 24 h. However, nitrate and nitrite levels in Choachi strain-inoculated hemolymph remained low, and this parasite strain multiplied rapidly, especially following peak superoxide activity, and eventually invaded the salivary glands for transmission to a vertebrate host. In both strains, short form epimastigotes stimulated greater superoxide and prophenoloxidase responses than long form epimastigotes. Injection of the NADPH oxidase inhibitor N-ethylmaleimide or the inducible nitric oxide synthase inhibitor S-methyl isothiourea sulfate caused significantly higher insect mortalities in groups of R. prolixus inoculated with either parasite strain compared with those of uninfected control insects. This indicates that both NADPH oxidase and nitric oxide synthase activity may be involved in the immune response of R. prolixus to infection by T. rangeli. Finally, Western blotting of R. prolixus hemocyte lysates revealed the presence of a protein immunologically related to the human NADPH oxidase complex, the initiator enzyme of the respiratory burst.     

The distribution of agglutinins and lytic activity against Trypanosoma rangeli and erythrocytes in Rhodnius prolixus and Triatoma infestans tissue extracts and haemolymph.

Haemolymph, heads, salivary glands, crops, midguts, hindguts, and Malpighian tubules from Rhodnius prolixus and Triatoma infestans were extracted in phosphate or Tris buffer saline with calcium, and tested for agglutination and lytic activities by microtitration against both vertebrate erythrocytes and cultured epimastigote forms of Trypanosoma rangeli. Haemagglutination activity against rabbit erythrocytes was found in the crop, midgut and hindgut extracts of T. infestans but only in the haemolymph of R. prolixus. Higher titers of parasite agglutinins were found in R. prolixus haemolymph than T. infestans, whilst the converse occurred for the tissue extracts. In addition, the extracts of T. infestans salivary glands, but not those of R. prolixus, showed a trypanolytic activity that was heat-inactivated and was not abolished by pre-incubation with any of the sugars or glycoproteins tested. T. infestans, which is refractory to infection by T. rangeli, thus appears to contain a much wider distribution of agglutinating and trypanolytic factors in its tissues than the more susceptible species, R. prolixus.     

Studies on Trypanosoma rangeli Tejera, 1920. IX. Course of infection in different stages of Rhodnius prolixus

Frequent individual observations od different stages of Rhodnius prolixus exposed to Trypanosoma rangeli, revealed a higher susceptibility to infection in the bugs exposed during the two first instars. The mortality rate in infected bugs was significantly higher than in controls, indicating that the parasite was responsible for the majority of deaths. An analysis of the mortality distribution, per instar, is presented. Statistical analysis of deaths among the different infected instars, showed that T. rangeli produces its pathological effect in any stage of R. prolixus independently of its susceptibility to the parasite. The survival to adult decreased in all the infected instar bugs. A significant longer time to reach the adult stage was observed in the infected bugs when compared with controls, excepting for specimens exposed in the third instar. The epidemiological significance of the present results is discussed.     

Effects of eicosanoid biosynthesis inhibitors on the prophenoloxidase-activating system and microaggregation reactions in the hemolymph of Rhodnius prolixus infected with Trypanosoma rangeli

Investigations on the effects of eicosanoid biosynthesis inhibitors on the hemocyte microaggregation and prophenoloxidase (proPO)-activating system in the hemolymph, parasitemia and mortality of Rhodnius prolixus infected with Trypanosoma rangeli were performed. Hemocoelic injection of live T. rangeli epimastigotes into fifth-instar larvae of R. prolixus that previously fed on blood containing an inhibitor of phospholipase A(2) (dexamethasone), a specific inhibitor of the cyclooxygenase pathway (indomethacin), and a non-selective lipoxygenase inhibitor (NDGA) (i) reduced the hemocyte microaggregation, (ii) attenuated the proPO system in the hemolymph and (iii) enhanced parasitemia and mortality induced by the parasite challenge in these insects. The effects obtained by dexamethasone administered orally were counteracted by inoculation of the insects with arachidonic acid. We suggest that the infectivity of T. rangeli can be increased by interference with the R. prolixus immune system. This is the first demonstration that the triatomine's immune responses to a parasite infection are modulated by a physiological system that includes eicosanoid biosynthesis.