Role of the head in the ultrastructural midgut organization in Rhodnius prolixus larvae: evidence from head transplantation experiments and ecdysone therapy

Studies on the effects of decapitation, head transplantation and ecdysone therapy on the ultrastructural organization of the midgut in 5th-instar larvae of Rhodnius prolixus, were carried out. Control insects had a typical and significant organization of the epithelial cells (mainly microvilli, extracellular membrane layers and basal portion of the epithelial cells) of the midgut (stomach and intestine) during the entire period of the experiment. However, the host larvae, when decapitated 1 day after feeding, demonstrated significant changes in the ultrastructural organization of the epithelial cells of these compartments. In converse experiments, head transplantations from untreated donors 4-5 days after feeding into headless larvae sustained the ultrastructural organization of the epithelial cells in the midgut. Oral therapy with ecdysone (5 &mgr;g/mL of blood meal) in decapitated insects significantly reversed the altered organization of the stomach and intestine. These results point to a brain factor, possibly the prothoracicotropic hormone (PTTH) which stimulates ecdysteroid production in the prothoracic glands, may be a factor responsible, directly or indirectly, for the midgut cell organization in R. prolixus.     

Trypanosoma cruzi: effects of azadirachtin and ecdysone on the dynamic development in Rhodnius prolixus larvae

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.     

Antiserum against perimicrovillar membranes and midgut tissue reduces the development of Trypanosoma cruzi in the insect vector, Rhodnius prolixus

Antiserum raised against Rhodnius prolixus perimicrovillar membranes (PMM) and midgut tissue interfered with the midgut structural organization and reduced the development of Trypanosoma cruzi in the R. prolixus insect vector. SDS-PAGE and Western blot analyses confirmed the specific recognition of midgut proteins by the antibody. Feeding, mortality, molt, and oviposition of the insects were unaffected by feeding with the antiserum. However, the eclosion of the eggs were reduced from R. prolixus females treated with antiserum. Additionally, in vivo evaluation showed that after oral treatment with the antiserum, the intensity of infection with the Dm-28c clone of T. cruzi decreased in the digestive tract of fifth-instar nymphs and in the excretions of R. prolixus adults. These results suggest that the changes observed in the PMM organization in the posterior midgut of R. prolixus may not be important for triatomine survival but the antiserum acts as a transmission-reduction vaccine able to induce significant decreases in T. cruzi infection in the vector.     

Trypanosoma cruzi: attachment to perimicrovillar membrane glycoproteins of Rhodnius prolixus

Studies were carried out to identify proteins involved in the interface of Trypanosoma cruzi with the perimicrovillar membranes (PMM) of Rhodnius prolixus. Video microscopy experiments demonstrated high level of adhesion of T. cruzi Dm 28c epimastigotes to the surface of posterior midgut cells of non-treated R. prolixus. The parasites however were unable to attach to gut cells obtained from decapitated or azadirachtin-treated insects. The influence of carbohydrates on the adhesion to insect midgut was confirmed by inhibition of parasite attachment after midgut incubation with N-acetylgalactosamine, N-acetylmannosamine, N-acetylglucosamine, D-galactose, D-mannose or sialic acid. We observed that hydrophobic proteins in the surface of epimastigotes bind to polypeptides with 47.7, 45.5, 44, 43, 40.5, 36, 31 and 13kDa from R. prolixus PMM and that pre-incubation of lectins specifically inhibited binding to 31, 40.5, 44 and 45.5kDa proteins. We suggest that glycoproteins from PMM and hydrophobic proteins from epimastigotes are important for the adhesion of the parasite to the posterior midgut cells of the vector.     

Effect of azadirachtin on the development of Trypanosoma cruzi in different species of triatomine insect vectors: long-term and comparative studies.

Studies were carried out on the effects of azadirachtin on the course of Trypanosoma cruzi infection in the gut of different triatomine vector species. In Rhodinus prolixus the development of T. cruzi clone Dm28c decreased in a dose-dependent manner, and the ED50 of this inhibitory effect was 0.25 microgram azadirachtin/ml bloodmeal. Using this insect, we conducted a long-term experiment which showed that azadirachtin (1.0 microgram/ml bloodmeal) completely blocks the development of T. cruzi even 120 days after treatment with the drug, and after four infectant meals. Similarly, the elimination of T. cruzi in feces and urine was also completely blocked over a period of 50 days after infection in insects treated with azadirachtin. Fifth-instar larvae of R. prolixus, Triatoma infestans, and Dipetalogaster maximus, infected with different clone/strains of T. cruzi, displayed drastic inhibition of trypanosome development when treated with azadirachtin. The discussion of these results focuses on the possibility that azadirachtin may act directly on gut physiology and/or indirectly through the neurosecretory system.     

Effect of blood components, abdominal distension, and ecdysone therapy on the ultrastructural organization of posterior midgut epithelial cells and perimicrovillar membranes in Rhodnius prolixus

The effects of blood components, nerve-cord severance, and ecdysone therapy on the posterior midgut epithelial cells of 5th-instar Rhodnius prolixus nymphs 10 days after feeding were analyzed by transmission electron microscopy. Cutting the nerve-cord of the blood-fed insects partially reduced the development of microvilli and perimicrovillar membranes (PMM), and produced large vacuoles and small electrondense granules; insects fed on Ringer's saline diet exhibited well developed microvilli and low PMM production; swolled rough endoplasmatic reticulum and electrondense granules; Ringer's saline meal with ecdysone led to PMM development, glycogen particles, and several mitochondria in the cytoplasm; epithelial cells of the insects fed on Ringer's saline meal whose nerve-cord was severed showed heterogeneously distributed microvilli with reduced PMM production and a great quantity of mitochondria and glycogen in the cytoplasm; well developed microvilli and PMM were observed in nerve-cord severed insects fed on Ringer's saline meal with ecdysone; Ringer's saline diet containing hemoglobin recovered the release of PMM; and insects fed on human plasma showed slightly reduced PMM production, although the addition of ecdysone in the plasma led to a normal midgut ultrastructural organization. We suggest that the full development of microvilli and PMM in the epithelial cells depends on the abdominal distension in addition to ingestion of hemoglobin, and the release of ecdysone.     

Cellular immune response in Rhodnius prolixus: role of ecdysone in hemocyte phagocytosis

In this paper we investigate in vivo and in vitro effects of orally administered azadirachtin and ecdysone on the phagocytic responses of Rhodnius prolixus 5th-instar larval hemocytes to the yeast Saccharomyces cerevisiae. Groups of insects fed non-treated blood (control) and insects that received azadirachtin plus ecdysone in the blood meal were inoculated with yeast cells in the hemocele. The injected yeast cells disappeared rapidly from the hemolymph, being removed completely by 90min after inoculation. In the insects treated only with azadirachtin the clearance of free yeast circulating particles was significantly delayed compared to the two previously mentioned groups. It was demonstrated that the binding of yeast cells to hemocytes was reduced in the insects treated only with azadirachtin in comparison to both non-treated control and azadirachtin plus ecdysone-treated groups. Phagocytosis occurred when yeast cells were added to hemocyte monolayers prepared with hemolymph from blood fed insects, treated or not with azadirachtin plus ecdysone, so that yeast cells were rapidly bound to hemocytes and internalized in high numbers. By contrast, insects treated with azadirachtin exhibited a drastic reduction in the quantity of yeast cell-hemocyte binding and subsequent internalization. In all groups, the hemocytes attached to the glass slides were predominantly plasmatocytes. The magnitude and speed of the cellular response suggests that hemocyte phagocytosis is one of the main driving forces for the clearance of free circulating yeast cells from the hemolymph. We propose that ecdysone modulates phagocytosis in R. prolixus larvae, and that this effect is antagonized by azadirachtin.     

Trypanosoma cruzi: effects of infection on cathepsin D activity in the midgut of Rhodnius prolixus

Cathepsin D activity was estimated in midgut homogenates from Rhodnius prolixus, uninfected and experimentally infected with Trypanosoma cruzi, at different times after blood ingestion. No enzyme activity was found in the anterior midgut and rectum. In the posterior midgut, enzyme activity was found both in lumen and wall. In starved uninfected insects, in lumen and wall, cathepsin D activity was high, decreasing to a constant rate at 1-15 days after feeding. In insects infected with T. cruzi cathepsin D activity increased 1 and 3 days after blood meal. We suggest that these changes in cathepsin D activity in R. prolixus posterior midgut are due to the establishment of T. cruzi infection.     

Trypanosoma cruzi immune response modulation decreases microbiota in Rhodnius prolixus gut and is crucial for parasite survival and development

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.     

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.     

Effects of azadirachtin on insect cytochrome P-450 dependent ecdysone 20-monooxygenase activity

The effects of the insect growth and ecdysis inhibitor azadirachtin on ecdysone 20-monooxygenase activity were examined in three insect species. Homogenates of wandering stage third instar larvae of Drosophila melanogaster, or abdomens from adult female Aedes aegypti, or fat body or midgut from last instar larvae of Manduca sexta were incubated with radiolabelled ecdysone and increasing concentrations of azadirachtin and the ecdysone 20-monoxygenase activity quantified by radioassay. Azadirachtin was found to inhibit in a dose-response fashion the ecdysone 20-monooxygenase activity associated with all the insect preparations. The concentration of azadirachtin required to elicit approximately 50% inhibition of the ecdysone 20-monooxygenase activity ranged from a low of 1 x 10(-4) M for Drosophila to a high of 4 x 10(-4) M for Manduca midgut.     

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.     

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.     

Trypanosoma cruzi heparin-binding proteins mediate the adherence of epimastigotes to the midgut epithelial cells of Rhodnius prolixus

Heparin-binding proteins (HBPs) have been demonstrated in both infective forms of Trypanosoma cruzi and are involved in the recognition and invasion of mammalian cells. In this study, we evaluated the potential biological function of these proteins during the parasite-vector interaction. HBPs, with molecular masses of 65·8 kDa and 59 kDa, were isolated from epimastigotes by heparin affinity chromatography and identified by biotin-conjugated sulfated glycosaminoglycans (GAGs). Surface plasmon resonance biosensor analysis demonstrated stable receptor-ligand binding based on the association and dissociation values. Pre-incubation of epimastigotes with GAGs led to an inhibition of parasite binding to immobilized heparin. Competition assays were performed to evaluate the role of the HBP-GAG interaction in the recognition and adhesion of epimastigotes to midgut epithelial cells of Rhodnius prolixus. Epithelial cells pre-incubated with HBPs yielded a 3·8-fold inhibition in the adhesion of epimastigotes. The pre-treatment of epimastigotes with heparin, heparan sulfate and chondroitin sulfate significantly inhibited parasite adhesion to midgut epithelial cells, which was confirmed by scanning electron microscopy. We provide evidence that heparin-binding proteins are found on the surface of T. cruzi epimastigotes and demonstrate their key role in the recognition of sulfated GAGs on the surface of midgut epithelial cells of the insect vector.     

Experimental transmission of the parasitic flagellates Trypanosoma cruzi and Trypanosoma rangeli between triatomine bugs or mice and captive neotropical bats

Trypanosoma cruzi and Trypanosoma rangeli-like trypanosomes have been found in a variety of neotropical bat species. In this study, bats (Artibeus lituratus, Carollia perspicillata, Desmodus rotundus, Glossophaga soricina, Molossus molossus, Phyllostomus hastatus) were maintained under controlled conditions, and experiments were conducted to determine how they might become infected naturally with trypanosomes. All bats were first screened for existing infections by hemoculture and the examination of blood smears, and only apparently uninfected animals were then used in the experiments. Proof was obtained that the triatomine bug Rhodnius prolixus would readily feed upon some of the bats, and two species became infected after being bitten by bugs infected with T. rangeli. Some bats also became infected by ingesting R. prolixus carrying T. cruzi, or following subcutaneous or intragastic inoculation with fecal suspensions of R. prolixus containing T. cruzi. P. hastatus became infected after ingesting mice carrying T. cruzi. All of the bats studied inhabit roosts that may be occupied by triatomine bugs and, with the exception of D. rotundus, all also feed to at least some extent upon insects. These findings provide further evidence of how bats may play significant roles in the epidemiology of T. cruzi and T. rangeli in the New World tropics.     

Developmental stages of Trypanosoma cruzi-like flagellates in Cavernicola pilosa

The developmental stages of Trypanosoma cruzi ssp., found in the intestinal tract of Cavernicola pilosa, are described and measurements given for nine life stages. The frequencies of the various stages in foregut, midgut and hindgut of the triatomines are provided; parasites were rare in the foregut and metatrypomastigotes were seen only in the mid- and hindguts. All adult bugs examined harboured intestinal infections of T. cruzi-like flagellates, large clumps of amastigotes were frequently observed in the midgut. The faeces of C. pilosa, containing metacyclic trypomastigotes, did not produce patent parasitaemia when inoculated into mice. Inoculated mice were not protected against subsequent challenge infections with the highly virulent Tulahuen stock of T. c. cruzi. The blood of bats also failed to produce parasitaemia when inoculated into mice, nor were the mice protected against subsequent challenges with T. c. cruzi. Although the developmental stages described were very similar to those of T. c. cruzi it is presumed that they were stages of T. c. marinkellei because of their failure to infect mice and Rhodnius prolixus, and their failure to protect inoculated mice against challenge with T. c. cruzi.     

Phenol oxidases from Rhodnius prolixus: Temporal and tissue expression pattern and regulation by ecdysone

Rhodnius prolixus 5th instar nymphs have significant PO enzymatic activity in the anterior midgut, fat body and hemolymph. The tissue with the major amount of PO activity is the anterior midgut while those with higher specific activities are the fat body and hemolymph. In this work the temporal pattern of PO enzymatic activity in different tissues was investigated. In fat body, PO peaks occur at 7, 12 and 16 days after a blood meal. In hemolymph, PO diminishes until day 7, and then recovers by day 14. In the anterior midgut tissue, PO peaks on day 9 and just before ecdysis; a similar pattern was observed in the anterior midgut contents. Some of these activities are dependent on the release of ecdysone, as feeding blood meal containing azadirachtin suppresses them and ecdysone treatment counteracts this effect. These results suggest that during the development of the 5th instar, the insect has natural regulating cycles of basal PO expression and activation, which could be related to the occurrence of natural infections. The differences in temporal patterns of activity and the effects of azadirachtin and ecdysone in each organ suggest that, at least in R. prolixus, different tissues are expressing different PO genes.     

Development of symbionts in triatomine bugs and the effects of infections with trypanosomatids

In the intestinal tract of fifth instars of the hematophagous reduviid bugs Rhodnius prolixus and Triatoma infestans blood ingestion induced an initial decrease of the concentration of the respective symbiotic bacteria Rhodococcus rhodnii and Nocardia sp. and then within 10 days a 15- or 18-fold increase of the total population/bug to about 0.8 x 10(9) colony-forming units in R. prolixus and 1.8 x 10(9) colony-forming units in T. infestans. About 95-99% of the total populations of both symbionts developed in the anterior midgut regions, i.e., cardia and stomach. The passage from the blood-storing stomach to the digesting small intestine caused a considerable breakdown of symbiont populations, and only about 0.01% of the total population was present in the rectum. These were excreted mainly within 4 h after a blood meal. After infection with three species of trypanosomatids, R. rhodnii, the symbiont of R. prolixus, was affected by Trypanosoma rangeli, but not by Blastocrithidia triatomae or Trypanosoma cruzi. On the other hand, in T. infestans the concentration of Nocardia sp. was reduced after infection with B. triatomae, but not by T. rangeli nor T. cruzi. In long-term B. triatomae-infected T. infestans, this reduction and a reduced diuretic activity after feeding synergistically lowered the symbiont concentration in the singly deposited feces/urine drops drastically compared to uninfected controls. These data strongly support the theory of the mechanisms of pathogenicity of T. rangeli and B. triatomae for R. prolixus and T. infestans, respectively, that the primal point of attack is the host-specific symbiont, R. rhodnii and Nocardia sp., respectively.     

Exploring the role of insect host factors in the dynamics of Trypanosoma cruzi-Rhodnius prolixus interactions

Members of the subfamily Triatominae, family Reduviidae, comprise a large number of insect species of which some are vectors of Trypanosoma cruzi, the causative agent of Chagas' disease. This article outlines research on the process of transformation and the dynamics of developmental stages of Trypanosoma cruzi in the triatomine insect hosts. Special attention is given to the interactions of parasites with gut molecules, and the gut environment, and with host developmental physiology and intestinal organization. The vector insect's permissiveness to Trypanosoma cruzi, which develops in the vector gut, largely depends on the host nutritional state, the parasite strain, trypanolytic compounds, digestive enzymes, lectins, resident bacteria in the gut and the endocrine system of the insect vector. Finally, the mechanisms of these interactions and their significance for Trypanosoma cruzi transmission are discussed.