Knowledge on the Rhodnius immune system and its activation in response to microorganism infections has grown in recent years. The first defences against microbiological infections are the structural barriers outside or inside the body (for example, exoskeleton and the perimicrovillar membrane in the midgut [55, 57–59].
The establishment of T. rangeli infection in both gut and hemocele of the insect vector is possibly regulated by a range of biochemical and physiological processes. The first environment for the transformation and development of T. rangeli is in the gut. There the parasites are confronted with anterior and posterior midgut components and products of blood digestion. These included bacteria [60, 61], hemolytic factors  and lectins [46, 63], all of which may modulate the infection of T. rangeli in the vector gut.
Once in the hemocele, T. rangeli must overcome the robust insect vector's defence system including lysozymes and trypanolytic activities , PPO activation , phagocytosis and hemocyte microaggregate formations [29, 30, 65–67], agglutination [46, 63], superoxide and nitric oxide production  and a trypanolytic protein which acts specifically against the T. rangeli KP1-strains . All these activities seem to act as biological barriers raising difficulties for the development and transmission of the parasite in the vector.
Humoral reactions and T. rangeli infection
Although Lopez et al.  showed that defensin was induced both in the hemolymph and midgut of R. prolixus by inoculation of Escherichia coli and Microccocus luteus, there are some data on the inactivation of the R. prolixus humoral immune system by parasite infection. Mello et al.  demonstrated that, after systemic inoculation of T. rangeli short epimastigotes into the hemocele of R. prolixus, the parasite produces a high intensity of infection through successive division during the extracellular development, with a concomitant increased levels in the lysozyme activity in the hemolymph. They also showed that T. rangeli infection induced neither trypanolytic nor peptide antibacterial activities, but a galactose-binding lectin from R. prolixus hemolymph, which enhanced the activation of clump formation by T. rangeli in R. prolixus hemocyte monolayers. An increase in clump size and hemocyte aggregation was also described . This purified lectin also affected in vitro the motility and survival of T. rangeli culture short forms, but not the long forms , which are predominant in the hemolymph two days after inoculation .
Another important biological event of T. rangeli interference in the insect immune reactions is its ability to activate the PPO system of R. prolixus. Gregorio and Ratcliffe [71, 72] demonstrated that Triatoma infestans, but not R. prolixus, presents a very active PPO system when activated by laminarin and lipopolysaccharides. For both species of insects, neither T. rangeli from culture nor parasite lysates were able to trigger PPO activation in vitro. However, the presence of the parasite in R. prolixus hemolymph assays reduced the level of PPO activation by laminarin. These authors suggest that the susceptibility of R. prolixus to T. rangeli hemolymph infection may, at least in part, be explained by the suppression of the inset immune defence system i.e. inhibition of the PPO cascade in the presence of this parasite.
Interestingly, Gomes et al.  clearly demonstrated using in vitro experiments that the activation of the PPO pathway occurred when the hemolymph was incubated with fat body homogenates and short epimastigote forms of T. rangeli. The same authors using in vivo experiments showed that short, but not long, epimastigote forms activated directly the formation of melanin . In addition, the PPO-activating pathway was suppressed when insects, which had been fed on blood containing either short or long epimastigotes, were challenged by thoracic inoculation of the short forms. This indicates that the reduction of the PO activity was a result of parasite ingestion. The PPO pathway is activated when glycosylphosphatidylinositol (GPI) anchors, specifically glycoinositolphospholipids (GIPLs) and GPI-mucins purified from T. rangeli epimastigotes, are inoculated in the insect .
One factor that can be important for killing T. rangeli is nitric oxide and nitrite/nitrate radicals, products of NO synthase (NOS) activity. Whitten et al.  described experiments to demonstrate whether or not nitric oxide and superoxide production could operate during T. rangeli infection in R. prolixus. These authors followed the inoculation of two strains and two developmental forms of T. rangeli after 24 h. When the H14 strain was inoculated, the parasites failed to multiply and invade the salivary glands whilst the Choachi strain rapidly multiplied in the hemolymph to invade salivary glands. However, in insects inoculated with H14 strain, the levels of PPO and superoxide generated by R. prolixus were significantly higher than Choachi strain, and nitrite and nitrate levels were also much higher with H14 inoculations. Usually, short forms of epimastigotes stimulated greater superoxide and PPO reactions than long epimastigotes in both parasite strains in the hemolymph of R. prolixus. Furthermore, when the NADPH oxidase inhibitor, N-ethylmaleimide, or the inhibitor of the inducible nitric oxide synthase, S-methylisothiourea sulfamide, are injected into R. prolixus, they resulted in higher insect mortality after T. rangeli infection of either strains compared with those untreated controls . Whitten et al.  demonstrated that the most pronounced reactions to crude LPS occurred in the R. prolixus fat body and hemocytes, while tissues of the digestive tract were most responsive to infections by T. cruzi and T. rangeli. This suggests that the NO-mediated immune responses in this insect are pathogen specific and independently modified both at the transcriptional and NO synthase gene expression.
It is interesting to note that in a screening of R. prolixus genes activated after T. cruzi infection by sequencing of subtractive libraries, no genes related to the humoral immune response were found to be transcriptionally upregulated . These results suggest that the R. prolixus immune responses to parasites are not mediated by AMPs, and could be centered in hemocytes nodulation, encapsulation and phagocytosis. The comparison between the responses against bacteria and T. cruzi also showed that R. prolixus activates different mechanisms of defence depending on the pathogen . In this way, it is possible that the regulation of immune related genes in R. prolixus differs significantly after T. cruzi or T. rangeli infection.
Hemocyte microaggregation and phagocytosis and T. rangeli infection: role of eicosanoids and PAF pathways
The circulating hemocytes are essential for the insect immunity. In R. prolixus seven morphological hemocyte types were identified by phase-contrast microscopy: prohemocytes, granulocytes, plasmatocytes, cystocytes, oenocytes and adipohemocytes and giant cells . Some cellular immune reactions have been studied in T. rangeli-triatomine interactions. Garcia et al. , demonstrated for the first time that eicosanoid biosynthesis inhibitors applied to R. prolixus strongly affect hemocyte microaggregation, one of the cellular immune reactions. The main data found by these authors were: (i) insects that had previously been fed on blood containing biosynthesis inhibitors of PLA2 (dexamethasone) and COX (indomethacin) and non-selective LOX inhibitor (nordihydroguaiaretic acid, NDGA) showed a significant increase in the number of free epimastigote forms of T. rangeli in the hemolymph and, consequently, increased lethality; and (ii) the parasite infection in insects treated with these compounds led to less hemocyte microaggregation and attenuated the activation of PPO system in the hemolymph.
Garcia et al.  suggest that arachidonic acid was not available in insects treated with dexamethasone. Indeed, the application of arachidonic acid significantly enhanced both hemocyte microaggregation and PO activity in the hemolymph of insects previously treated with dexamethasone and challenged with parasites. It also reduced the number of parasites in the circulation and the mortality of insects. The effects of indomethacin and NDGA were considered relevant because they indicated the influence of multiple eicosanoid metabolites in immune reactions of R. prolixus infected with T. rangeli. Furthermore, hemocelic inoculation of epimastigotes of T. rangeli into larvae of R. prolixus previously fed with blood containing the same parasite, demonstrated a reduced number of hemocyte microaggregates, enhanced the number of parasites in the hemolymph as well as increased the mortality of these insects. All these effects were counteracted by combined injection of R. prolixus with T. rangeli and arachidonic acid . These results suggest that the 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 (Figure 3).
One interesting novelty of this parasite-vector interaction was revealed by Machado et al. . They demonstrated that hemocelic injection of short T. rangeli epimastigotes in R. prolixus that were previously fed with blood containing WEB 2086 [a strong platelet-activating factor (2-acetyl-1-hexadecyl-sn-glycero-3-phosphocholine (PAF) antagonist] resulted in reduced hemocyte microaggregation, attenuated PPO activation in the hemolymph as well as increased the parasitemia and insect mortality. Nevertheless, simultaneous application of PAF did not counteract hemocytes microaggregation and PO activity.
It was demonstrated that physalin B, a natural secosteroidal chemical from Physalis angulata, induces immunodepression in R. prolixus [80–82] and strongly blocks hemocyte phagocytosis and microaggregate formations in R. prolixus . The inhibition induced by physalin B was counteracted for both phagocytosis and microaggregation of hemocytes by arachidonic acid or PAF applied by hemocelic injection. Physalin B did not alter hemocyte PLA2 activities but it significantly enhanced PAF-acetyl hydrolase (PAF-AH) activity in the cell free hemolymph and hemocytes. Theses findings reinforce the importance of PAF and arachidonic acid pathways in cellular immune reactions in R. prolixus (Figure 3).
The most exciting outcome in the investigation of T. rangeli in triatomines is the PAF influence on the hemocyte nodulation  and phagocytic responses of R. prolixus hemocytes against Saccharomyces cerevisiae [66, 67]. These authors evaluated the effects of PAF and eicosanoids in the phagocytosis in hemocyte monolayers (the main cell type implicated in this process is plasmatocytes) of R. prolixus against the yeast S. cerevisiae. The experiments demonstrated that the phagocytosis of yeast cells by Rhodnius hemocytes is very efficient in both controls and cells treated with PAF or arachidonic acid. However, phagocytosis of yeast particles is significantly diminished when the specific inhibitor of PLA2, dexamethasone, is applied to the hemocytes. By contrast, dexamethasone pre-treated hemocyte monolayers exhibit a drastic enhancement in the quantity of yeast cell-hemocyte internalizations when the cells are treated with arachidonic acid. Phagocytosis decreases expressively in hemocyte monolayers treated with WEB 2086, a specific PAF receptor antagonist. Nevertheless, a decrease of phagocytosis with WEB 2086 is also counteracted by the treatment with PAF [66, 67]. The authors suggest that these data on phagocytosis of yeast cells by hemocytes are related to the activation of PAF receptors and provides a novel insight into the cell signaling pathway of non-self recognition related to cellular immune reactions in the insect-parasite relationship.
Finally, Figueiredo et al.  demonstrated that hemocyte phagocytosis was significantly reduced by oral infection with T. rangeli. These authors demonstrated that hemocyte phagocytosis inhibition caused by the parasite infection was rescued by exogenous arachidonic acid or PAF applied by hemocelic injection. They also observed an attenuation of PLA2 activities in R. prolixus hemocytes (cytosolic PLA2: cPLA2, secreted PLA2: sPLA2 and Ca++-independent PLA2: iPLA2) and an increase of sPLA2 in cell-free hemolymph. At the same time, the PAF-AH activity in the cell-free hemolymph enhanced considerably. These data suggest that T. rangeli infection depresses eicosanoids and insect PAF analogous (iPAF) pathways giving support to the role of PLA2 in the modulation of arachidonic acid and iPAF biosynthesis and of PAF-acetylhydrolase (PAF-AH) by reducing the concentration of iPAF in R. prolixus . The relationship between the expression of the genes of PLA2 and PAF-AH as well as general cellular responses and signal transduction pathways is poorly understood in hemipterans. In this way, it is difficult to interpret the T. rangeli immunosuppression in terms of regulation of cellular signal transduction cascades. The data above suggest an inhibition of the NF-κB pathway, one well known effect of physalin treatment in mammal cells . This is in agreement with the inhibition of the humoral immune response, but more detailed studies on the molecular mechanisms are needed to clarify this point.
All these finding illustrate the ability of T. rangeli to modulate the cellular immune responses of R. prolixus to favor its own multiplication in the hemolymph.