Influence of Serratia marcescens and Rhodococcus rhodnii on the Humoral Immunity of Rhodnius prolixus.

Chagas disease is a human infectious disease caused by Trypanosoma cruzi and can be transmitted by triatomine vectors, such as Rhodnius prolixus. One limiting factor for T. cruzi development is the composition of the bacterial gut microbiota in the triatomine. Herein, we analyzed the humoral immune responses of R. prolixus nymphs treated with antibiotics and subsequently recolonized with either Serratia marcescens or Rhodococcus rhodnii. The treatment with antibiotics reduced the bacterial load in the digestive tract, and the recolonization with each bacterium was successfully detected seven days after treatment. The antibiotic-treated insects, recolonized with S. marcescens, presented reduced antibacterial activity against Staphylococcus aureus and phenoloxidase activity in hemolymph, and lower nitric oxide synthase (NOS) and higher defensin C gene (DefC) gene expression in the fat body. These insects also presented a higher expression of DefC, lower prolixicin (Prol), and lower NOS levels in the anterior midgut. However, the antibiotic-treated insects recolonized with R. rhodnii had increased antibacterial activity against Escherichia coli and lower activity against S. aureus, higher phenoloxidase activity in hemolymph, and lower NOS expression in the fat body. In the anterior midgut, these insects presented higher NOS, defensin A (DefA) and DefC expression, and lower Prol expression. The R. prolixus immune modulation by these two bacteria was observed not only in the midgut, but also systemically in the fat body, and may be crucial for the development and transmission of the parasites Trypanosoma cruzi and Trypanosoma rangeli.

Nitric oxide effects on Rhodnius prolixus's immune responses, gut microbiota and Trypanosoma cruzi development.

The immune system of Rhodnius prolixus comprehends the synthesis of different effectors that modulate the intestinal microbiota population and the life cycle of the parasite Trypanosoma cruzi inside the vector midgut. One of these immune responses is the production of reactive nitrogen species (RNS) derived by the action of nitric oxide synthase (NOS). Therefore, we investigated the effects of L-arginine, the substrate for nitric oxide (NO) production and Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME), an inhibitor of NOS, added in the insect blood meal. We analyzed the impact of these treatments on the immune responses and development of intestinal bacteria and parasites on R. prolixus nymphs. The L-arginine treatment in R. prolixus nymphs induced a higher NOS gene expression in the fat body and increased NO production, but reduced catalase and antimicrobial activities in the midgut. As expected, L-NAME treatment reduced NOS gene expression in the fat body. In addition, L-NAME treatment diminished catalase activity in the hemolymph and posterior midgut reduced phenoloxidase activity in the anterior midgut and increased the antimicrobial activity in the hemolymph. Both treatments caused a reduction in the cultivatable intestinal microbiota, especially in insects treated with L-NAME. However, T. cruzi development in the insect's digestive tract was suppressed after L-arginine treatment and the opposite was observed with L-NAME, which resulted in higher parasite counts. Therefore, we conclude that induction and inhibition of NOS and NO production are associated with other R. prolixus humoral immune responses, such as catalase, phenoloxidase, and antibacterial activities in different insect organs. These alterations reflect on intestinal microbiota and T. cruzi development.

Rhodnius prolixus: Identification of missing components of the IMD immune signaling pathway and functional characterization of its role in eliminating bacteria.

The innate immune system in insects is regulated by specific signalling pathways. Most immune related pathways were identified and characterized in holometabolous insects such as Drosophila melanogaster, and it was assumed they would be highly conserved in all insects. The hemimetabolous insect, Rhodnius prolixus, has served as a model to study basic insect physiology, but also is a major vector of the human parasite, Trypanosoma cruzi, that causes 10,000 deaths annually. The publication of the R. prolixus genome revealed that one of the main immune pathways, the Immune-deficiency pathway (IMD), was incomplete and probably non-functional, an observation shared with other hemimetabolous insects including the pea aphid (Acyrthosiphon pisum) and the bedbug (Cimex lectularius). It was proposed that the IMD pathway is inactive in R. prolixus as an adaptation to prevent eliminating beneficial symbiont gut bacteria. We used bioinformatic analyses based on reciprocal BLAST and HMM-profile searches to find orthologs for most of the "missing" elements of the IMD pathway and provide data that these are regulated in response to infection with Gram-negative bacteria. We used RNAi strategies to demonstrate the role of the IMD pathway in regulating the expression of specific antimicrobial peptides (AMPs) in the fat body of R. prolixus. The data indicate that the IMD pathway is present and active in R. prolixus, which opens up new avenues of research on R. prolixus-T. cruzi interactions.

Comparative genomics analysis of triatomines reveals common first line and inducible immunity-related genes and the absence of Imd canonical components among hemimetabolous arthropods.

BACKGROUND: Insects operate complex humoral and cellular immune strategies to fend against invading microorganisms. The majority of these have been characterized in Drosophila and other dipterans. Information on hemipterans, including Triatominae vectors of Chagas disease remains incomplete and fractionated. RESULTS: We identified putative immune-related homologs of three Triatominae vectors of Chagas disease, Triatoma pallidipennis, T. dimidiata and T. infestans (TTTs), using comparative transcriptomics based on established immune response gene references, in conjunction with the predicted proteomes of Rhodnius prolixus, Cimex lecticularis and Acyrthosiphon pisum hemimetabolous. We present a compressive description of the humoral and cellular innate immune components of these TTTs and extend the immune information of other related hemipterans. Key homologs of the constitutive and induced immunity genes were identified in all the studied hemipterans. CONCLUSIONS: Our results in the TTTs extend previous observations in other hemipterans lacking several components of the Imd signaling pathway. Comparison with other hexapods, using published data, revealed that the absence of various Imd canonical components is common in several hemimetabolous species.

Rhodnius prolixus: from physiology by Wigglesworth to recent studies of immune system modulation by Trypanosoma cruzi and Trypanosoma rangeli.

This review is dedicated to the memory of Professor Sir Vincent B. Wigglesworth (VW) in recognition of his many pioneering contributions to insect physiology which, even today, form the basis of modern-day research in this field. Insects not only make vital contributions to our everyday lives by their roles in pollination, balancing eco-systems and provision of honey and silk products, but they are also outstanding models for studying the pathogenicity of microorganisms and the functioning of innate immunity in humans. In this overview, the immune system of the triatomine bug, Rhodnius prolixus, is considered which is most appropriate to this dedication as this insect species was the favourite subject of VW's research. Herein are described recent developments in knowledge of the functioning of the R. prolixus immune system. Thus, the roles of the cellular defences, such as phagocytosis and nodule formation, as well as the role of eicosanoids, ecdysone, antimicrobial peptides, reactive oxygen and nitrogen radicals, and the gut microbiota in the immune response of R. prolixus are described. The details of many of these were unknown to VW although his work gives indications of his awareness of the importance to R. prolixus of cellular immunity, antibacterial activity, prophenoloxidase and the gut microbiota. This description of R. prolixus immunity forms a backdrop to studies on the interaction of the parasitic flagellates, Trypanosoma cruzi and Trypanosoma rangeli, with the host defences of this important insect vector. These parasites remarkably utilize different strategies to avoid/modulate the triatomine immune response in order to survive in the extremely hostile host environments present in the vector gut and haemocoel. Much recent information has also been gleaned on the remarkable diversity of the immune system in the R. prolixus gut and its interaction with trypanosome parasites. This new data is reviewed and gaps in our knowledge of R. prolixus immunity are identified as subjects for future endeavours. Finally, the publication of the T. cruzi, T. rangeli and R. prolixus genomes, together with the use of modern molecular techniques, should lead to the enhanced identification of the determinants of infection derived from both the vector and the parasites which, in turn, could form targets for new molecular-based control strategies.

Humoral and cellular immune responses induced by the urease-derived peptide Jaburetox in the model organism Rhodnius prolixus.

BACKGROUND: Although the entomotoxicity of plant ureases has been reported almost 20 years ago, their insecticidal mechanism of action is still not well understood. Jaburetox is a recombinant peptide derived from one of the isoforms of Canavalia ensiformis (Jack Bean) urease that presents biotechnological interest since it is toxic to insects of different orders. Previous studies of our group using the Chagas disease vector and model insect Rhodnius prolixus showed that the treatment with Jack Bean Urease (JBU) led to hemocyte aggregation and hemolymph darkening, among other effects. In this work, we employed cell biology and biochemical approaches to investigate whether Jaburetox would induce not only cellular but also humoral immune responses in this species. RESULTS: The findings indicated that nanomolar doses of Jaburetox triggered cation-dependent, in vitro aggregation of hemocytes of fifth-instar nymphs and adults. The use of specific eicosanoid synthesis inhibitors revealed that the cellular immune response required cyclooxygenase products since indomethacin prevented the Jaburetox-dependent aggregation whereas baicalein and esculetin (inhibitors of the lipoxygenases pathway) did not. Cultured hemocytes incubated with Jaburetox for 24 h showed cytoskeleton disorganization, chromatin condensation and were positive for activated caspase 3, an apoptosis marker, although their phagocytic activity remained unchanged. Finally, in vivo treatments by injection of Jaburetox induced both a cellular response, as observed by hemocyte aggregation, and a humoral response, as seen by the increase of spontaneous phenoloxidase activity, a key enzyme involved in melanization and defense. On the other hand, the humoral response elicited by Jaburetox injections did not lead to an increment of antibacterial or lysozyme activities. Jaburetox injections also impaired the clearance of the pathogenic bacteria Staphylococcus aureus from the hemolymph leading to increased mortality, indicating a possible immunosuppression induced by treatment with the peptide. CONCLUSIONS: In our experimental conditions and as part of its toxic action, Jaburetox activates some responses of the immune system of R. prolixus both in vivo and in vitro, although this induction does not protect the insects against posterior bacterial infections. Taken together, these findings contribute to the general knowledge of insect immunity and shed light on Jaburetox's mechanism of action.

Impact of Trypanosoma cruzi on antimicrobial peptide gene expression and activity in the fat body and midgut of Rhodnius prolixus.

BACKGROUND: Rhodnius prolixus is a major vector of Trypanosoma cruzi, the causative agent of Chagas disease in Latin America. In natural habitats, these insects are in contact with a variety of bacteria, fungi, virus and parasites that they acquire from both their environments and the blood of their hosts. Microorganism ingestion may trigger the synthesis of humoral immune factors, including antimicrobial peptides (AMPs). The objective of this study was to compare the expression levels of AMPs (defensins and prolixicin) in the different midgut compartments and the fat body of R. prolixus infected with different T. cruzi strains. The T. cruzi Dm 28c clone (TcI) successfully develops whereas Y strain (TcII) does not complete its life- cycle in R. prolixus. The relative AMP gene expressions were evaluated in the insect midgut and fat body infected on different days with the T. cruzi Dm 28c clone and the Y strain. The influence of the antibacterial activity on the intestinal microbiota was taken into account. METHODS: The presence of T. cruzi in the midgut of R. prolixus was analysed by optical microscope. The relative expression of the antimicrobial peptides encoding genes defensin (defA, defB, defC) and prolixicin (prol) was quantified by RT-qPCR. The antimicrobial activity of the AMPs against Staphylococcus aureus, Escherichia coli and Serratia marcescens were evaluated in vitro using turbidimetric tests with haemolymph, anterior and posterior midgut samples. Midgut bacteria were quantified using colony forming unit (CFU) assays and real time quantitative polymerase chain reaction (RT-qPCR). RESULTS: Our results showed that the infection of R. prolixus by the two different T. cruzi strains exhibited different temporal AMP induction profiles in the anterior and posterior midgut. Insects infected with T. cruzi Dm 28c exhibited an increase in defC and prol transcripts and a simultaneous reduction in the midgut cultivable bacteria population, Serratia marcescens and Rhodococcus rhodnii. In contrast, the T. cruzi Y strain neither induced AMP gene expression in the gut nor reduced the number of colony formation units in the anterior midgut. Beside the induction of a local immune response in the midgut after feeding R. prolixus with T. cruzi, a simultaneous systemic response was also detected in the fat body. CONCLUSIONS: R. prolixus AMP gene expressions and the cultivable midgut bacterial microbiota were modulated in distinct patterns, which depend on the T. cruzi genotype used for infection.

Rhodnius prolixus interaction with Trypanosoma rangeli: modulation of the immune system and microbiota population.

BACKGROUND: Trypanosoma rangeli is a protozoan that infects a variety of mammalian hosts, including humans. Its main insect vector is Rhodnius prolixus and is found in several Latin American countries. The R. prolixus vector competence depends on the T. rangeli strain and the molecular interactions, as well as the insect's immune responses in the gut and haemocoel. This work focuses on the modulation of the humoral immune responses of the midgut of R. prolixus infected with T. rangeli Macias strain, considering the influence of the parasite on the intestinal microbiota. METHODS: The population density of T. rangeli Macias strain was analysed in different R. prolixus midgut compartments in long and short-term experiments. Cultivable and non-cultivable midgut bacteria were investigated by colony forming unit (CFU) assays and by 454 pyrosequencing of the 16S rRNA gene, respectively. The modulation of R. prolixus immune responses was studied by analysis of the antimicrobial activity in vitro against different bacteria using turbidimetric tests, the abundance of mRNAs encoding antimicrobial peptides (AMPs) defensin (DefA, DefB, DefC), prolixicin (Prol) and lysozymes (LysA, LysB) by RT-PCR and analysis of the phenoloxidase (PO) activity. RESULTS: Our results showed that T. rangeli successfully colonized R. prolixus midgut altering the microbiota population and the immune responses as follows: 1 - reduced cultivable midgut bacteria; 2 - decreased the number of sequences of the Enterococcaceae but increased those of the Burkholderiaceae family; the families Nocardiaceae, Enterobacteriaceae and Mycobacteriaceae encountered in control and infected insects remained the same; 3 - enhanced midgut antibacterial activities against Serratia marcescens and Staphylococcus aureus; 4 - down-regulated LysB and Prol mRNA levels; altered DefB, DefC and LysA depending on the infection (short and long-term); 5 - decreased PO activity. CONCLUSION: Our findings suggest that T. rangeli Macias strain modulates R. prolixus immune system and modifies the natural microbiota composition.

Imunidade humoral de Rhodnius prolixus: impacto sobre a microbiota e desenvolvimento de Trypanosoma cruzi e Trypanosoma rangeli / Humoral immunity of Rhodnius prolixus: impact on microbiota and Trypanosoma cruzi and Trypanosoma rangeli development

Rhodnius prolixus é um dos principais insetos vetores de Trypanosomacruzi e de Trypanosoma rangeli na América Latina. A produção de peptídeosantimicrobianos (AMPs) no trato digestivo ou corpo gorduroso do inseto é vitalpara evitar a proliferação de microrganismos patogênicos além de manter ahomeostasia da microbiota nativa. O presente trabalho focou na modulação daimunidade humoral do intestino médio de R. prolixus desafiados oralmente coma bactéria Gram-positiva Staphylococcus aureus e Gram-negativa Escherichiacoli, além de seus tripanosomatídeos naturais T. rangeli e T. cruzi, considerandoa influência do desenvolvimento dos parasitas sobre a microbiota intestinal. Emcondições normais, a região anterior do intestino médio houve maior abundânciade transcritos de genes de lisozimas (lis) e defensinas (def), enquanto naposterior, do gene da prolixicina (prol). Insetos alimentados com bactérias Gramnegativasapresentaram maior quantidade de transcritos de defC e prol,enquanto a ingestão de bactérias Gram-positivas induziu a expressão de defA edefB no intestino médio. A infecção por T. rangeli cepa Macias diminuiu aatividade fenoloxidásica, os níveis de expressão de lisozimas e prolixicina, aomesmo tempo em que induziu aumento de atividade antibacteriana e dos níveisde defensina C no tubo digestivo do inseto, também modificando a composiçãode bactérias nativas...

Rhodnius prolixus is a major vector of Trypanosoma rangeli andTrypanosoma cruzi, in Latin America. The production of antimicrobial peptides(AMPs) in the midgut of the insect is vital to control possible infection, and tomaintain the microbiota already present in the digestive tract. This work focuseson the modulation of the humoral immune responses of the midgut of R. prolixusorally challenged with Gram positive and Gram negative bacteria as well with T.rangeli Macias strain, T. cruzi Dm 28c and Y strain, considering the influence ofthe parasites on the intestinal microbiota. Our results showed that the anteriormidgut contents of control insects contain a higher inducible antibacterial activityand AMPs transcript abundance than those of the posterior midgut. Insects orallyfed with Gram-negative bacteria presented higher amount of defC and proltranscripts, while the ingestion of Gram-positive induced defA and defBexpression in the midgut. T. rangeli Macias strain successfully colonized R.prolixus midgut through a decreasing in PO activities, prolixicin and lysozymelevels, while at the same time induced an increase in antibacterial activity andupregulated defC levels in the insect anterior midgut. T. rangeli infection alsodiminishes the amount of cultivable gut bacteria as well modified the compositionof indigenous microorganisms. Furthermore, different T. cruzi strains presentdistinct profiles of immune system and microbiota modulation in R. prolixusmidgut, where T. cruzi Dm 28c was able to induce an increase in defensin Cgenes and a depression in prolixicin genes, while drastically reduce the cultivablebacteria population. In the other hand T. cruzi Y was not competent to induceAMPs expression in the gut or considerably reduce the microbiota in the anteriormidgut...

Humoral responses in Rhodnius prolixus: bacterial feeding induces differential patterns of antibacterial activity and enhances mRNA levels of antimicrobial peptides in the midgut.

BACKGROUND: The triatomine, Rhodnius prolixus, is a major vector of Trypanosoma cruzi, the causative agent of Chagas disease in Latin America. It has a strictly blood-sucking habit in all life stages, ingesting large amounts of blood from vertebrate hosts from which it can acquire pathogenic microorganisms. In this context, the production of antimicrobial peptides (AMPs) in the midgut of the insect is vital to control possible infection, and to maintain the microbiota already present in the digestive tract. METHODS: In the present work, we studied the antimicrobial activity of the Rhodnius prolixus midgut in vitro against the Gram-negative and Gram-positive bacteria Escherichia coli and Staphylococcus aureus, respectively. We also analysed the abundance of mRNAs encoding for defensins, prolixicin and lysozymes in the midgut of insects orally infected by these bacteria at 1 and 7 days after feeding. RESULTS: Our results showed that the anterior midgut contents contain a higher inducible antibacterial activity than those of the posterior midgut. We observed that the main AMP encoding mRNAs in the anterior midgut, 7 days after a blood meal, were for lysozyme A, B, defensin C and prolixicin while in the posterior midgut lysozyme B and prolixicin transcripts predominated. CONCLUSION: Our findings suggest that R. prolixus modulates AMP gene expression upon ingestion of bacteria with patterns that are distinct and dependent upon the species of bacteria responsible for infection.

Physalin B inhibits Trypanosoma cruzi infection in the gut of Rhodnius prolixus by affecting the immune system and microbiota.

Physalin B is a natural secosteroidal, extracted from the Solanaceae plant, Physalis angulata, and it presents immune-modulator effects on the bloodsucking bug, Rhodnius prolixus. In this work, R. prolixus was treated with physalin B at a concentration of 1 mg/ml of blood meal (oral application), or 20 ng/insect (applied topically) or 57 ng/cm(2) of filter paper (contact treatment), and infected with Trypanosoma cruzi Dm28c clone (2×10(6) epimastigotes/insect). The three types of applications significantly decreased the number of T. cruzi Dm28c in the gut comparing with the non-treated infected insects (controls). All groups of infected insects treated with physalin B had higher numbers of bacterial microbiota in the gut than the non-treated controls infected with T. cruzi. We observed that the infected physalin B insects with topical and contact treatments had a lower antibacterial activity in the gut when compared with control infected insects. Furthermore, infected insects with the physalin B oral treatment produced higher levels of nitrite and nitrate in the gut than control infected insects. These results demonstrate that physalin B decreases the T. cruzi transmission by inhibiting the parasite development in the insect vector R. prolixus. Herein the importance of physalin B modulation on the immune system and microbiota population in terms of parasite development and transmission are discussed.

Prolixicin: a novel antimicrobial peptide isolated from Rhodnius prolixus with differential activity against bacteria and Trypanosoma cruzi.

We identified and characterized the activity of prolixicin, a novel antimicrobial peptide (AMP) isolated from the hemipteran insect, Rhodnius prolixus. Sequence analysis reveals one region of prolixicin that may be related to the diptericin/attacin family of AMPs. Prolixicin is an 11-kDa peptide containing a putative 21 amino acid signal peptide, two putative phosphorylation sites and no glycosylation sites. It is produced by both adult fat body and midgut tissues in response to bacterial infection of the haemolymph or the midgut. Unlike most insect antibacterial peptides, the prolixicin gene does not seem to be regulated by NF-κB binding sites, but its promoter region contains several GATA sites. Recombinant prolixicin has strong activity against the Gram-negative bacterium Escherichia coli and differential activity against several Gram-negative and Gram-positive bacteria. No significant toxicity was demonstrated against Trypanosoma cruzi, the human parasite transmitted by R. prolixus.

Microscopic and molecular characterization of ovarian follicle atresia in Rhodnius prolixus Stahl under immune challenge.

In this work we characterized the degenerative process of ovarian follicles of the bug Rhodnius prolixus challenged with the non-entomopathogenic fungus Aspergillus niger. An injection of A. niger conidia directly into the hemocoel of adult R. prolixus females at the onset of vitellogenesis caused no effect on host lifespan but elicited a net reduction in egg batch size. Direct inspection of ovaries from the mycosed insects revealed that fungal challenge led to atresia of the vitellogenic follicles. Light microscopy and DAPI staining showed follicle shrinkage, ooplasm alteration and disorganization of the monolayer of follicle cells in the atretic follicles. Transmission electron microscopy of thin sections of follicle epithelium also showed nuclei with condensed chromatin, electron dense mitochondria and large autophagic vacuoles. Occurrence of apoptosis of follicle cells in these follicles was visualized by TUNEL labeling. Resorption of the yolk involved an increase in protease activities (aspartyl and cysteinyl proteases) which were associated with precocious acidification of yolk granules and degradation of yolk protein content. The role of follicle atresia in nonspecific host-pathogen associations and the origin of protease activity that led to yolk resorption are discussed.

Physalin B inhibits Rhodnius prolixus hemocyte phagocytosis and microaggregation by the activation of endogenous PAF-acetyl hydrolase activities.

The effects of physalin B (a natural secosteroidal chemical from Physalis angulata, Solanaceae) on phagocytosis and microaggregation by hemocytes of 5th-instar larvae of Rhodnius prolixus were investigated. In this insect, hemocyte phagocytosis and microaggregation are known to be induced by the platelet-activating factor (PAF) or arachidonic acid (AA) and regulated by phospholipase A(2) (PLA(2)) and PAF-acetyl hydrolase (PAF-AH) activities. Phagocytic activity and formation of hemocyte microaggregates by Rhodnius hemocytes were strongly blocked by oral treatment of this insect with physalin B (1mug/mL of blood meal). The inhibition induced by physalin B was reversed for both phagocytosis and microaggregation by exogenous arachidonic acid (10microg/insect) or PAF (1microg/insect) applied by hemocelic injection. Following treatment with physalin B there were no significant alterations in PLA(2) activities, but a significant enhancement of PAF-AH was observed. These results show that physalin B inhibits hemocytic activity by depressing insect PAF analogous (iPAF) levels in hemolymph and confirm the role of PAF-AH in the cellular immune reactions in R. prolixus.

Arrest of oogenesis in the bug Rhodnius prolixus challenged with the fungus Aspergillus niger is mediated by immune response-derived PGE2.

In this work we characterized the immune response of the insect Rhodnius prolixus to a direct injection into the hemocoel of the non-entomopathogenic fungus Aspergillus niger, and evaluated its consequences on host oogenesis. These animals were able to respond by mounting effective cellular and humoral responses to this fungus; these responses were shown, however, to have reproductive fitness costs, as the number of eggs laid per female was significantly reduced. The disturbance of egg formation during infectious process correlated with an elevation in the titer of hemolymph prostaglandin E2 48 h post-challenge. Administration of Zymosan A as an immunogenic non-infectious challenge produced similar effects on phenoloxidase and prophenoloxidase activities, oocyte development and prostaglandin E2 titer, precluding the hypothesis of an effect mediated by fungal metabolites in animals challenged with fungus. Ovaries at 48 h post-challenge showed absence of vitellogenic ovarian follicles, and the in vivo administration of prostaglandin E2 or its receptor agonist misoprostol, partially reproduced this phenotype. Together these data led us to hypothesize that immune-derived prostaglandin E2 raised from the insect response to the fungal challenge is involved in disturbing follicle development, contributing to a reduction in host reproductive output and acting as a host-derived adaptive effector to infection.

Characterization of neuropeptide F-like immunoreactivity in the blood-feeding hemipteran, Rhodnius prolixus.

The invertebrate neuropeptide Y (NPY) homolog, neuropeptide F (NPF), has been characterized for a wide range of invertebrate phyla, including platyhelminthes, molluscs, and arthropods. Current hypotheses suggest that NPF may be capable of regulating responses to diverse external cues related to nutritional status and feeding. The qualitative and quantitative distribution of an NPF-like peptide in fifth instar Rhodnius prolixus was undertaken using an antiserum raised against Drosophila NPF. Immunohistochemistry reveals NPF-like immunoreactive neurons and processes in the central nervous system, stomatogastric nervous system and peripheral nervous system. The distribution of NPF-like immunoreactivity within the medial neurosecretory cells of the brain and neurohemal areas of the corpus cardiacum and dorsal vessel, suggests NPF may act as a neurohormone. Immunoreactive processes are present over the surface of the hindgut and the immunoreactivity in these processes is greatly reduced in intensity 24h post-feeding. The quantification of partially purified NPF-like material in the CNS of R. prolixus was conducted by HPLC fractionation and radioimmunoassay. The results suggest that NPF-like material is present in fifth instar R. prolixus and likely released into the hemolymph following a blood meal.

Differential modulation of Rhodnius prolixus nitric oxide activities following challenge with Trypanosoma rangeli, T. cruzi and bacterial cell wall components.

Nitric oxide (NO) is a key immune effector and signaling molecule in many organisms. However, the contribution NO makes towards insect immunity has received little attention, particularly in non-dipteran species. In this study, tissue- and time-specific alterations in NO synthase (NOS) gene expression and NO production were documented in the hemipteran vector of Chagas' disease, Rhodnius prolixus, following in vivo immune challenge by Trypanosoma cruzi, T. rangeli and crude bacterial lipopolysaccharide (LPS). The most pronounced reactions to crude LPS occurred in major immune tissues (fat body and blood cells), while tissues of the digestive tract were most responsive to infections by T. cruzi and T. rangeli. The data suggest the NO-mediated immune responses of R. prolixus are pathogen-specific and independently modified both at the transcriptional and enzyme levels.

Rhodnius prolixus: identification of immune-related genes up-regulated in response to pathogens and parasites using suppressive subtractive hybridization.

We report the identification of immune-related molecules from the fat body, and intestine of Rhodnius prolixus, an important vector of Chagas disease. Insects were challenged by introducing pathogens or Trypanosoma cruzi, the parasite that causes Chagas disease, into the hemocoel. RNA from intestines, or fat body were isolated 24h after stimulation. We used suppressive subtractive hybridization to identify immune-related genes, generated three subtracted libraries, sequenced the clones and assembled the sequences. The functional annotation revealed expressed sequence tags (ESTs) generated in response to various stimuli in all tissues, and included pathogen recognition molecules, regulatory molecules, and effector molecules.

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.

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.