An updated catalog of lipocalins of the chagas disease vector Rhodnius prolixus (Hemiptera, Reduviidae).

The haematophagy process by arthropods has been one of the main targets of studies in the parasite-host interaction, and the kissing-bug Rhodnius prolixus, vector of the protozoan Trypanosoma cruzi, has been one of the main models for such studies. Still in the 1980s, it was identified that among the salivary proteins for disrupting vertebrate host homeostasis, lipocalins were among the most relevant proteins for this process. Since then, 30 lipocalins have been identified in the salivary glands of R. prolixus, that promotes vasodilatation, prevents inflammation, act as anticoagulants and inhibits platelet aggregation. The present work aims to identify new lipocalins from R. prolixus, combining transcriptome and genome data. Identified new genes were mapped and had their structure annotated. To infer an evolutionary relationship between lipocalins, and to support the predicted functions for each lipocalin, all amino acid sequences were used to construct phylogenetic trees. We identified a total of 29 new lipocalins, 3 new bioaminogenic-biding proteins (which act to inhibit platelet aggregation and vasodilation), 9 new inhibitors of platelet aggregation, 7 new apolipoproteins and 10 lipocalins with no putative function. In addition, we observed that several of the lipocalins are also expressed in different R. prolxius tissues, including gut, central nervous system, antennae, and reproductive organs. In addition to newly identified lipocalins and a mapping the new and old lipocalins in the genome of R. prolixus, our study also carried out a review on functional status and nomenclature of some of the already identified lipocalins. Our study reinforces that we are far from understanding the role of lipocalins in the physiology of R. prolixus, and that studies of this family are still of great relevance.

Effect of Triatoma infestans saliva on mouse immune system cells: The role of the pore-forming salivary protein trialysin.

Triatoma infestans is one of the most important vectors of Trypanosoma cruzi in the Americas. While feeding, they release large amounts of saliva that will counteract the host's responses triggered at the bite site. Despite the various activities described on T. infestans saliva, little is known about its effect on the modulation of the host's immune system. This work aimed to describe the effects of T. infestans saliva on cells of the mouse immune system and access the role in hematophagy. The effect of saliva or salivary gland extract (SGE) was evaluated in vivo and in vitro by direct T. infestans feeding on mice or using different biological assays. Mice that were submitted to four bites by three specimens of T. infestans had their anti-saliva IgG serum levels approximately 2.4 times higher than controls, but no change in serum IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-γ, and TNF-α levels was observed. No macroscopic alterations were seen at the bite site, but an accumulation of mononuclear and polymorphonuclear cells shortly after the bite and 24 h later were observed in histological cuts. At low concentrations (up to ∼5 µg/well), SGE induced TNF-α production by macrophages and spleen cells, IFN-γ and IL-10 by spleen cells and NO by macrophages. However, at higher concentrations (10 and 20 µg/well), viability of macrophages and spleen cells was reduced by SGE, reducing the production of NO and cytokines (except TNF-α). The salivary trialysin was the main inducer of cell death as macrophage viability and NO production was restored in assays carried out with SGE from trialysin knockdown insects. The reduction of the salivary trialysin by RNAi affected the total ingestion rate, the weight gain, and retarded the molt from second to the fifth instar of T. infestans nymphs fed on mice. The results show that T. infestans saliva modulates the activity of cells of the host immune system and trialysin is an important salivary molecule that reduces host cells viability and impacts the feeding performance of T. infestans feeding on live hosts.

Locomotion activity and its effects on the survival of Amblyomma sculptum (Acari: Ixodidae) nymphs under laboratory conditions.

The active locomotion of ticks is directly associated with the epidemiology of tick-borne diseases, as it has important implications for the interaction of ticks with their hosts and their dispersion in the environment. In an attempt to elucidate the factors involved in the dispersion of Amblyomma sculptum, the present work aimed to characterize different aspects of the active locomotion of A. sculptum nymphs under laboratory conditions. To this end, nymphs were placed on a string at a 70° inclination and their walking activity was recorded daily along with their survival period. During their lifetime, ticks walked an average of 110 m. Their locomotion was not in a straight line and nymphs changed direction 142 times throughout their lifetimes. The mean distance walked per experimental day was 1.8 m, while the average walking distance before changing direction was 52 cm. The distance walked per experimental day reduced over time. The survival of ticks was affected by walking; resting nymphs survived for over 6 months, while the survival of those that walked daily was reduced to approximately 62 days. The results showed that A. sculptum nymphs were able to cover distances of over 100 m throughout their lifetimes, but they walked short distances at a time and constantly changed direction. This behavior indicates that ticks are not able to disperse over long distances by means of active locomotion.

Amblyomma sculptum Salivary Protease Inhibitors as Potential Anti-Tick Vaccines.

Amblyomma sculptum is the main tick associated with human bites in Brazil and the main vector of Rickettsia rickettsii, the causative agent of the most severe form of Brazilian spotted fever. Molecules produced in the salivary glands are directly related to feeding success and vector competence. In the present study, we identified sequences of A. sculptum salivary proteins that may be involved in hematophagy and selected three proteins that underwent functional characterization and evaluation as vaccine antigens. Among the three proteins selected, one contained a Kunitz_bovine pancreatic trypsin inhibitor domain (named AsKunitz) and the other two belonged to the 8.9 kDa and basic tail families of tick salivary proteins (named As8.9kDa and AsBasicTail). Expression of the messenger RNA (mRNA) encoding all three proteins was detected in the larvae, nymphs, and females at basal levels in unfed ticks and the expression levels increased after the start of feeding. Recombinant proteins rAs8.9kDa and rAsBasicTail inhibited the enzymatic activity of factor Xa, thrombin, and trypsin, whereas rAsKunitz inhibited only thrombin activity. All three recombinant proteins inhibited the hemolysis of both the classical and alternative pathways; this is the first description of tick members of the Kunitz and 8.9kDa families being inhibitors of the classical complement pathway. Mice immunization with recombinant proteins caused efficacies against A. sculptum females from 59.4% with rAsBasicTail immunization to more than 85% by immunization with rAsKunitz and rAs8.9kDa. The mortality of nymphs fed on immunized mice reached 70-100%. Therefore, all three proteins are potential antigens with the possibility of becoming a new tool in the control of A. sculptum.

RNA-seq analysis of the salivary glands and midgut of the Argasid tick Ornithodoros rostratus.

Ornithodoros rostratus is a South American argasid tick which importance relies on its itchy bite and potential as disease vector. They feed on a wide variety of hosts and secrete different molecules in their saliva and intestinal content that counteract host defences and help to accommodate and metabolize the relatively large quantity of blood upon feeding. The present work describes the transcriptome profile of salivary gland (SG) and midgut (MG) of O. rostratus using Illumina sequencing. A total of 8,031 contigs were assembled and assigned to different functional classes. Secreted proteins were the most abundant in the SG and accounted for ~67% of all expressed transcripts with contigs with identity to lipocalins and acid tail proteins being the most representative. On the other hand, immunity genes were upregulated in MG with a predominance of defensins and lysozymes. Only 10 transcripts in SG and 8 in MG represented ~30% of all RNA expressed in each tissue and one single contig (the acid tail protein ORN-9707) represented ~7% of all expressed contigs in SG. Results highlight the functional difference of each organ and identified the most expressed classes and contigs of O. rostratus SG and MG.

Competence of non-human primates to transmit Leishmania infantum to the invertebrate vector Lutzomyia longipalpis.

Leishmaniasis is a zoonotic disease of worldwide relevance. Visceral leishmaniasis is endemic in Brazil, where it is caused by Leishmania infantum with Lutzomyia longipalpis being the most important invertebrate vector. Non-human primates are susceptible to L. infantum infection. However, little is known about the role of these species as reservoirs. The aim of this study was to evaluate the transmissibility potential of visceral leishmaniasis by non-human primates through xenodiagnosis using the phlebotomine Lu. longipalpis as well as to identify phlebotomine species prevalent in the area where the primates were kept in captivity, and assess infection by Leishmania in captured phlebotomine specimens. Fifty two non-human primates kept in captivity in an endemic area for leishmaniasis were subjected to xenodiagnosis. All primates were serologically tested for detection of anti-Leishmania antibodies. Additionally, an anti-Lu. longipalpis saliva ELISA was performed. Sand flies fed on all animals were tested by qPCR to identify and quantify L. infantum promastigotes. Eight of the 52 non-human primates were positive by xenodiagnosis, including three Pan troglodytes, three Leontopithecus rosalia, one Sapajus apella, and one Miopithecus talapoin, with estimated numbers of promastigotes ranging from 5.67 to 1,181.93 per µg of DNA. Positive animals had higher levels of IgG anti-Lu. longipalpis saliva when compared to negative animals, prior to xenodiagnosis. Captive non-human primates are capable of infecting Lu. longipalpis with L. infantum. Our findings also demonstrate the relevance of non-human primates as sentinels to zoonotic diseases. Several phlebotomine species, including Lu. longipalpis, have been identified in the area where the primates were maintained, but only one pool of Lutzomyia lenti was infected with L. infantum. This study has implications for public health strategies and conservation medicine.

Occurrence of the Parasitic Fly Philornis torquans on Fledglings of the Rufous-Fronted Thornbird ( Phacellodomus rufifrons) in Southeast Brazil.

Philornis is a neotropical genus of muscid fly that interacts with birds and may affect the development and survival of the birds' offspring. Although Philornis is a relatively common parasite, there is a lack of information about Philornis hosts in several parts of the Americas. In this study, two nests of the Rufousfronted Thornbird ( Phacellodomus rufifrons) were collected in Pedro Leopoldo, southeast Brazil. The first contained four nestlings of advanced age (about 20 d old) and a recently emerged Philornis torquans female adult fly. The second nest contained three nestlings (less than 7 d old) and several Philornis torquans subcutaneous larvae. One of the nestlings was infested by 53 larvae, which had attacked several parts of its body and caused individual wounds containing 1 to more than 15 larvae. The length of the larvae ranged from 3 to 18 mm and only one was a second instar; the remaining 69 were third instars. The pupal period lasted 9-13 d. In total, 71 larvae were collected from the nest, with nestling parasitism varying from 7 to 53 larvae (mean- 23.7±25.5 larvae/nestling).

Salivary Gland Extract Modulates the Infection of Two Leishmania enriettii Strains by Interfering With Macrophage Differentiation in the Model of Cavia porcellus.

The subgenus Mundinia includes several Leishmania species that have human and veterinary importance. One of those members, Leishmania Mundinia enriettii was isolated from the guinea pig Cavia porcellus in the 1940s. Several histopathological studies have already been performed in this species in the absence of salivary gland extract (SGE), which are determinant and the early and future events of the infection. Our main hypothesis is that SGE could differentially modulate the course of the lesion and macrophage differentiation caused by avirulent and virulent L. enriettii strains. Here, the C. porcellus nasal region was infected using needles with two strains of L. enriettii (L88 and Cobaia) in the presence/absence of SGE and followed for 12 weeks. Those strains vary in terms of virulence, and their histopathological development was characterized. Some L88-infected animals could develop ulcerated/nodular lesions, whereas Cobaia strain developed non-ulcerated nodular lesions. Animals experimentally inoculated developed a protuberance and/or lesion after the 4th and 5th weeks of infection. Macroscopically, the size of lesion in L88-infected animals was smaller in the presence of SGE. Remarkable differences were detected microscopically in the presence of SGE for both strains. After the 6th and 7th weeks, L88-infected animals were heavily parasitized with an intense inflammatory profile bearing amastigotes and pro-inflammatory cells compared to those infected by Cobaia strain. Morphometry analysis revealed that L1+ macrophages were abundant in the L88 infection, but not in the Cobaia infection. In the presence of SGE, an increased CD163+ macrophage infiltrate by both strains was detected. Interestingly, this effect was more pronounced in Cobaia-infected animals. This study showed the role of SGE during the course of L. enriettii (strains L88 and Cobaia) infection and its role in modulating macrophage attraction to the lesion site. SGE decreased L1+ macrophages and this may favor an escaping mechanism for L88 parasites. On the other hand, in the presence of SGE, an increase in CD163+ cells during Cobaia infection may be important for its control. Although both strains healed at the end of the infection, the role of SGE was determinant for the kinetics of the immunopathological events in this dermotropic species.

Functional aspects of salivary nitric oxide synthase of Rhodnius prolixus (Hemiptera, Reduviidae) and nitric oxide trafficking at the vector-host interface.

Rhodnius prolixus expresses nitric oxide synthase (NOS) in the cytosol of the salivary gland (SG) cells. The NO produced is stored in the SG lumen bound to NO-carrier haemeproteins called nitrophorins (NPs). NPs bind tightly to NO in the acidic SG lumen, but release NO when the pH becomes high, e.g., at the host skin (pH~7.4). NO elicits potent and transient relaxation of vascular smooth muscle. Here, we investigated the role of salivary NO in the R. prolixus feeding behaviour and the salivary vasodilator activity of the host microcirculation. NOS knockdown in R. prolixus changed the SG colour, decreased the number of NO-loaded NPs and caused impairment of feeding performance. When salivary gland extracts (SGEs) were obtained from NOS- and NPs-knockdown insects and prepared in pH 5.0 solution and injected (i.v.) into mice via the tail vein, no vasodilation was observed, whereas SGEs from control insects caused long-term venodilation in the mouse skin. SGs disrupted directly in PBS (pH 7.4) containing BSA produced long-term vasodilation compared to the controls without BSA due to the possible formation of nitroso-albumin, suggesting that host serum albumin extends the NO half-life when NO is injected into the host skin by triatomine during their blood-feeding.

Virus-like Particle Display of the α-Gal Carbohydrate for Vaccination against Leishmania Infection.

Secreted and surface-displayed carbohydrates are essential for virulence and viability of many parasites, including for immune system evasion. We have identified the α-Gal trisaccharide epitope on the surface of the protozoan parasites Leishmania infantum and Leishmania amazonensis, the etiological agents of visceral and cutaneous leishmaniasis, respectively, with the latter bearing larger amounts of α-Gal than the former. A polyvalent α-Gal conjugate on the immunogenic Qß virus-like particle was tested as a vaccine against Leishmania infection in a C57BL/6 α-galactosyltransferase knockout mouse model, which mimics human hosts in producing high titers of anti-α-Gal antibodies. As expected, α-Gal-T knockout mice infected with promastigotes of both Leishmania species showed significantly lower parasite load in the liver and slightly decreased levels in the spleen, compared with wild-type mice. Vaccination with Qß-α-Gal nanoparticles protected the knockout mice against Leishmania challenge, eliminating the infection and proliferation of parasites in the liver and spleen as probed by qPCR. The α-Gal epitope may therefore be considered as a vaccine candidate to block human cutaneous and visceral leishmaniasis.

Functional evaluation of Heat Shock Proteins 70 (HSP70/HSC70) on Rhodnius prolixus (Hemiptera, Reduviidae) physiological responses associated with feeding and starvation.

Blood-sucking vectors must overcome thermal stress caused by intake of proportionally large amounts of warm blood from their hosts. In response to this, Heat Shock Proteins (HSPs) such as the widely studied HSP70 family (the inducible HSP70 and the cognate form HSC70, known for their role in preserving essential cellular functions) are rapidly up-regulated in their tissues. The triatomine Rhodnius prolixus is an important vector of Trypanosoma cruzi, the causative pathogen of Chagas' disease, and is also a model organism for studying insect biology and physiology. In this work, we observed that the expression of Rhodnius prolixus HSP70 was rapidly up-regulated in response to thermal shocks (0 °C and 40 °C) and also during the first hours after feeding on blood. HSP70/HSC70 RNAi knockdown elicited important alterations in R. prolixus physiological responses triggered by blood meal and starvation. HSP70/HSC70 knockdown insects showed lower resistance to prolonged starvation in comparison to appropriate controls, dying between 32 and 40 days after dsRNA injection. After blood feeding, the physiological effects of HSP70/HSC70 knockdown were more prominent and the insects died even earlier, within 14-20 days after feeding (21-27 days after dsRNA injection). These bugs showed impaired blood processing and digestion, reduced energetic metabolism and the midgut immune responses were compromised. Our findings suggest that HSP70/HSC70 depletion affected R. prolixus in starvation or fed conditions. After feeding, the arrival of blood in the digestive tract of knockdown insects fails to activate essential signaling pathways involved in blood processing, producing several alterations in their physiological processes enough to generate a premature death.

Saliva of Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) inhibits classical and alternative complement pathways.

BACKGROUND: Rhipicephalus (Boophilus) microplus is the main ectoparasite affecting livestock worldwide. For a successful parasitism, ticks need to evade several immune responses of their hosts, including the activation of the complement system. In spite of the importance of R. microplus, previous work only identified one salivary molecule that blocks the complement system. The current study describes complement inhibitory activities induced by R. microplus salivary components and mechanisms elicited by putative salivary proteins on both classical and alternative complement pathways. RESULTS: We found that R. microplus saliva from fully- and partially engorged females was able to inhibit both pathways. Saliva acts strongly at the initial steps of both complement activation pathways. In the classical pathway, the saliva blocked C4 cleavage, and hence, deposition of C4b on the activation surface, suggesting that the inhibition occurs at some point between C1q and C4. In the alternative pathway, saliva acts by binding to initial components of the cascade (C3b and properdin) thereby preventing the C3 convertase formation and reducing C3b production and deposition as well as cleavage of factor B. Saliva has no effect on formation or decay of the C6 to C8 components of the membrane attack complex. CONCLUSION: The saliva of R. microplus is able to inhibit the early steps of classical and alternative pathways of the complement system. Saliva acts by blocking C4 cleavage and deposition of C4b on the classical pathway activation surface and, in the alternative pathway, saliva bind to initial components of the cascade (C3b and properdin) thereby preventing the C3 convertase formation and the production and deposition of additional C3b.

An Inhibitor of the Alternative Pathway of Complement in Saliva of New World Anopheline Mosquitoes.

The complement system present in circulating blood is an effective mechanism of host defense, responsible for the killing of pathogens and the production of potent anaphylatoxins. Inhibitors of the complement system have been described in the saliva of hematophagous arthropods that are involved in the protection of digestive tissues against complement system-mediated damage. In this study, we describe albicin, a novel inhibitor of the alternative pathway of complement from the salivary glands of the malaria vector, Anopheles albimanus The inhibitor was purified from salivary gland homogenates by reverse-phase HPLC and identified by mass spectrometry as a small (13.4-kDa) protein related to the gSG7 protein of Anopheles gambiae and Anopheles stephensi Recombinant albicin was produced in Escherichia coli and found to potently inhibit lysis of rabbit erythrocytes in assays of the alternative pathway while having no inhibitory effect on the classical or lectin pathways. Albicin also inhibited the deposition of complement components on agarose-coated plates, although it could not remove previously bound components. Antisera produced against recombinant albicin recognized both the native and recombinant inhibitors and also blocked their activities in in vitro assays. Using surface plasmon resonance and enzymatic assays, we found that albicin binds and stabilizes the C3-convertase complex (C3bBb) formed on a properdin surface and inhibits the convertase activity of a reconstituted C3bBb complex in solution. The data indicate that albicin specifically recognizes the activated form of the complex, allowing more efficient inhibition by an inhibitor whose quantity is limited.

Inhibition of the classical pathway of the complement system by saliva of Amblyomma cajennense (Acari: Ixodidae).

Inhibition of the complement system during and after haematophagy is of utmost importance for tick success in feeding and tick development. The role of such inhibition is to minimise damage to the intestinal epithelium as well as avoiding inflammation and opsonisation of salivary molecules at the bite site. Despite its importance, the salivary anti-complement activity has been characterised only in species belonging to the Ixodes ricinus complex which saliva is able to inhibit the alternative and lectin pathways. Little is known about this activity in other species of the Ixodidae family. Thus, the aim of this study was to describe the inhibition of the classical pathway of the complement system by the saliva of Amblyomma cajennense at different stages of the haematophagy. The A. cajennense saliva and salivary gland extract (SGE) were able to inhibit the complement classical pathway through haemolytic assays with higher activity observed when saliva was used. The anti-complement activity is present in the salivary glands of starving females and also in females throughout the whole feeding process, with significant higher activity soon after tick detachment. The SGE activity from both females fed on mice or horses had no significant correlation (p > 0.05) with tick body weight. The pH found in the intestinal lumen of A. cajennense was 8.04 ± 0.08 and haemolytic assays performed at pH 8.0 showed activation of the classical pathway similarly to what occurs at pH 7.4. Consequently, inhibition could be necessary to protect the tick enterocytes. Indeed, the inhibition observed by SGE was higher in pH 8.0 in comparison to pH 7.4 reinforcing the role of saliva in protecting the intestinal cells. Further studies should be carried out in order to identify the inhibitor molecule and characterise its inhibition mechanism.

SALO, a novel classical pathway complement inhibitor from saliva of the sand fly Lutzomyia longipalpis.

Blood-feeding insects inject potent salivary components including complement inhibitors into their host's skin to acquire a blood meal. Sand fly saliva was shown to inhibit the classical pathway of complement; however, the molecular identity of the inhibitor remains unknown. Here, we identified SALO as the classical pathway complement inhibitor. SALO, an 11 kDa protein, has no homology to proteins of any other organism apart from New World sand flies. rSALO anti-complement activity has the same chromatographic properties as the Lu. longipalpis salivary gland homogenate (SGH)counterparts and anti-rSALO antibodies blocked the classical pathway complement activity of rSALO and SGH. Both rSALO and SGH inhibited C4b deposition and cleavage of C4. rSALO, however, did not inhibit the protease activity of C1s nor the enzymatic activity of factor Xa, uPA, thrombin, kallikrein, trypsin and plasmin. Importantly, rSALO did not inhibit the alternative or the lectin pathway of complement. In conclusion our data shows that SALO is a specific classical pathway complement inhibitor present in the saliva of Lu. longipalpis. Importantly, due to its small size and specificity, SALO may offer a therapeutic alternative for complement classical pathway-mediated pathogenic effects in human diseases.

Transmissibility of Leishmania infantum from maned wolves (Chrysocyon brachyurus) and bush dogs (Speothos venaticus) to Lutzomyia longipalpis.

Leishmania (Leishmania) infantum is the cause of visceral leishmaniasis in the Americas. The disease is transmitted mostly through the bite of the invertebrate vector, the phlebotomine Lutzomyia longipalpis in the New World. Although the domestic dog is considered the most important reservoir of the disease, other mammalian, including wildlife, are susceptible to infection. The goal of this study was to perform xenodiagnosis to evaluate the capacity of naturally infected maned wolves (Chrysocyon brachyurus) and bush dogs (Speothos venaticus) to transmit Leishmania infantum to female sand flies (L. longipalpis). Xenodiagnoses were performed in February and August, 2013, when 77.7% (three maned wolves and four bush dogs) or 100% of the animals were positive, respectively. However, parasite loads in the engorged sand flies was low (<200 promastigotes and <150.2 parasites/µg of DNA). No statistically significant differences were observed between the two species or the two time points (February and August). In conclusion, this study demonstrated that maned wolves (C. brachyurus) and bush dogs (S. venaticus) asymptomatically infected with L. infantum are capable of transmitting L. infantum to the invertebrate host L. longipalpis, although the parasite loads in engorged phlebotomines exposed to these animals were very low.

Long-term effects and parental RNAi in the blood feeder Rhodnius prolixus (Hemiptera; Reduviidae).

RNA interference (RNAi) has been widely employed as a useful alternative to study gene function in insects, including triatomine bugs. However, several aspects related to the RNAi mechanism and functioning are still unclear. The aim of this study is to investigate the persistence and the occurrence of systemic and parental RNAi in the triatomine bug Rhodnius prolixus. For such, the nitrophorins 1 to 4 (NP1-4), which are salivary hemeproteins, and the rhodniin, an intestinal protein, were used as targets for RNAi. The dsRNA for both molecules were injected separately into 3rd and 5th instar nymphs of R. prolixus and the knockdown (mRNA levels and phenotype) were progressively evaluated along several stages of the insect's life. We observed that the NP1-4 knockdown persisted for more than 7 months after the dsRNA injection, and at least 5 months in rhodniin knockdown, passing through various nymphal stages until the adult stage, without continuous input of dsRNA. The parental RNAi was successful from the dsRNA injection in 5th instar nymphs for both knockdown targets, when the RNAi effects (mRNA levels and phenotype) were observed at least in the 2nd instar nymphs of the F1 generation. However, the parental RNAi did not occur when the dsRNA was injected in the 3rd instars. The confirmation of the long persistence and parental transmission of RNAi in R. prolixus can improve and facilitate the utilization of this tool in insect functional genomic studies.

Application of RNA interference in triatomine (Hemiptera: Reduviidae) studies.

Triatomines (Hemiptera: Reduviidae) are obligate hematophagous insects. They are of medical importance because they are vectors of Trypanosoma cruzi, the causative agent of Chagas disease in the Americas. In recent years, the RNA interference (RNAi) technology has emerged as a practical and useful alternative means of studying gene function in insects, including triatomine bugs. RNAi research in triatomines is still in its early stages, several issues still need to be elucidated, including the description of the molecules involved in the RNAi machinery and aspects related to phenotype evaluation and persistence of the knockdown in different tissues and organs. This review considers recent applications of RNAi to triatomine research, describing the major methods that have been applied during the knockdown process such as the double-stranded RNA delivery mechanism (injection, microinjection, or ingestion) and the phenotype characterization (mRNA and target protein levels) in studies conducted with the intent to provide greater insights into the biology of these insects. In addition to the characterization of insect biomolecules, some with biopharmacological potential, RNAi may provide a new view of the interaction between triatomine and trypanosomatids, enabling the development of new measures for vector control and transmission of the parasite.

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, ß-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.

Carbohydrate digestion in Lutzomyia longipalpis' larvae (Diptera - Psychodidae).

Lutzomyia longipalpis is the principal species of phlebotomine incriminated as vector of Leishmania infantum, the etiological agent of visceral leishmaniasis in the Americas. Despite its importance as vector, almost nothing related to the larval biology, especially about its digestive system has been published. The objective of the present study was to obtain an overview of carbohydrate digestion by the larvae. Taking in account that phlebotomine larvae live in the soil rich in decaying materials and microorganisms we searched principally for enzymes capable to hydrolyze carbohydrates present in this kind of substrate. The principal carbohydrases encountered in the midgut were partially characterized. One of them is a α-amylase present in the anterior midgut. It is probably involved with the digestion of glycogen, the reserve carbohydrate of fungi. Two other especially active enzymes were present in the posterior midgut, a membrane bound α-glucosidase and a membrane bound trehalase. The first, complete the digestion of glycogen and the other probably acts in the digestion of trehalose, a carbohydrate usually encountered in microorganisms undergoing hydric stress. In a screening done with the use of p-nitrophenyl-derived substrates other less active enzymes were also observed in the midgut. A general view of carbohydrate digestion in L. longipalpis was presented. Our results indicate that soil microorganisms appear to be the main source of nutrients for the larvae.