Changes in salivary nitrophorin profile during the life cycle of the blood-sucking bug Rhodnius prolixus

The insect Rhodnius prolixus is a hematophagous hemipteran that has five nymphal instars. Fifth instar nymphs contain, in their salivary glands, four nitrophorins which have already been described in the literature (NP1, NP2, NP3 and NP4). Two new hemeproteins were isolated and partially characterized from first instar nymphs. NP2, that shows an anticoagulant activity, was also identified, but NP1, NP3 and NP4 were not found. As these new hemeproteins have amino-terminal sequences clearly homologous to already described nitrophorins and were capable of binding nitric oxide, they were named nitrophorins 5 and 6, although they showed an unusual Soret band at 412 nm. In each subsequent nymphal stage, a new nitrophorin emerges. In the second instar, NP4 comes into view, in the third instar NP1 appears, and NP3 is only found in fifth instar nymphs and adults, showing that the nitrophorin profile of R. prolixus saliva is stage-specific.     

7-Aminoactinomycin D for apoptosis staining in flow cytometry

All species of the genus Rhodnius have a characteristic red coloration in their salivary glands due to the presence of heme proteins. Some of these secreted proteins, known as nitrophorins (NPs), are responsible for many of the antihemostatic activities of Rhodnius saliva such as anticoagulant and antihistamine. Several NPs have been described (NP1-4 and NP7), where NP7 is the only one with affinity to phospholipid membranes. Computational prediction suggested that NP7 also has an extended N-terminal tail on signal peptide cleavage; however, the complementary DNA does not allow the determination of the exact site of signal peptidase cleavage. On the other hand, according to previous studies, the exact length of the N-terminus has important consequences for the nitric oxide binding properties of NP7. Here, a method was developed to select phospholipid membrane-attaching proteins from homogenized tissue for analysis by mass spectrometry. The method was used to determine the exact N-terminus of the ferriheme protein NP7 from homogenates of the salivary glands of 5th instar nymphal stages of Rhodnius prolixus.     

RNA interference of the salivary gland nitrophorin 2 in the triatomine bug Rhodnius prolixus (Hemiptera: Reduviidae) by dsRNA ingestion or injection

Mass sequencing of cDNA libraries from salivary glands of triatomines has resulted in the identification of many novel genes of unknown function. The aim of the present work was to develop a functional RNA interference (RNAi) technique for Rhodnius prolixus, which could be widely used for functional genomics studies in triatomine bugs. To this end, we investigated whether double-stranded RNA (dsRNA) can inhibit gene expression of R. prolixus salivary nitrophorin 2 (NP2) and what impact this might have on anticoagulant and apyrase activity in the saliva. dsRNA was introduced by two injections or by ingestion. RT-PCR of the salivary glands showed that injections of 15 microg of NP2 dsRNA in fourth-instar nymphs reduced gene expression by 75+/-14% and that feeding 1 microg/microL of NP2 dsRNA into second-instar nymphs (approx. 13 microg in total) reduced gene expression by 42+/-10%. Phenotype analysis showed that saliva of normal bugs prolonged plasma coagulation by about four-fold when compared to saliva of knockdown bugs. These results and the light color of the salivary gland content from some insects are consistent with the knockdown findings. The findings suggest that RNAi will prove a highly valuable functional genomics technique in triatomine bugs. The finding that feeding dsRNA can induce knockdown is novel for insects.     

Nitrophorin synthesis is modulated by protein kinase CK2

Rhodnius prolixus is a blood-sucking bug whose saliva contains a family of nitric oxide-carrying proteins named nitrophorins (NPs). Saliva is injected into the host bloodstream during insect feeding. Nitric oxide is then released from NPs and will act on vascular smooth muscle, promoting vasodilation. Epithelial cells of salivary glands then undergo a massive synthesis of antihemostatics including NPs which produces saliva for the next blood meal. Here, we demonstrate the transient activation of a protein kinase in the salivary glands of R. prolixus after a blood meal. Biochemical, immunological, and pharmacological assays were used to identify this enzyme as protein kinase CK2. CK2 is activated after a blood meal and decreases to basal levels when salivary gland refilling is resumed. Inhibition of CK2 blocked [(35)S]methionine incorporation into newly synthesized salivary gland proteins in cultured tissue. Dissected salivary glands were then incubated with the heme fluorescent analog palladium (II) mesoporphyrin IX (Pd-MP) in the presence of a selective cell-permeable CK2 inhibitor, TBB (4,5,6,7-tetrabromobenzotriazole). NP synthesis was quantified based on fluorescence of the Pd-MP group bound to the NP heme pocket. TBB dramatically blocked NP synthesis. Altogether, these data are the first demonstration to show that antihemostatic synthesis in a blood-sucking arthropods is under protein phosphorylation control.     

Contractions associated with the salivary glands of the blood-feeding bug, Rhodnius prolixus: evidence for both a neural and neurohormonal coordination

The salivary glands of the blood-feeding bug, Rhodnius prolixus, are composed of a single epithelial layer of binucleate cells and a double layer of visceral muscle cells surrounding a large secretory cavity. The saliva contains substances which counteract the hemostasis of the host, and injection of saliva into the host is an essential component of successful and efficient gorging.The muscles surrounding the salivary glands of Rhodnius are under polyneuronal control from the salivary nerve projecting out of the hypocerebral ganglion. The amplitude of contractions induced by neural stimulation is dependent upon both intensity and frequency of nerve stimulation.Serotonin and FMRFamide-related peptides (FaRPs) are delivered in the nerve supply to the salivary glands, and both classes of neuroactive chemicals increase frequency and amplitude of phasic contractions in a dose-dependent manner. A member of the FaRP myosuppressin subfamily, however, inhibits contractions. CRF-related and Leucokinin-like peptides are not delivered in the nerve supply but may be present in the hemolymph during feeding. Leucokinin 1 and Zoone DH (a CRF-related peptide) both induce a dose-dependent increase in basal tonus, with phasic contractions superimposed. Zoone DH is more active than Leucokinin 1. Factors are present in the CNS of Rhodnius which mimic the effects of serotonin and the stimulatory peptides.     

Purification and characterization of a lipid transfer particle in Rhodnius prolixus: phospholipid transfer

In this study we report the purification and characterization of a lipid transfer particle (LTP) from Rhodnius prolixus hemolymph, and its participation in phospholipid and diacylglycerol transfer processes. (3)H-diacylglycerol labeled low density lipophorin from Manduca sexta ((3)H-LDLp) was incubated with R. prolixus lipophorin (Lp) in the presence of Rhodnius hemolymph. Following incubation and isolation, both lipoproteins showed equivalent amounts of (3)H-labeled lipids. Hemolymph was subjected to KBr gradient ultracentrifugation. SDS-PAGE analysis of gradient fractions showed the enrichment of bands with molecular masses similar to the M. sexta LTP standard. LTP containing fractions were assayed and lipid transfer activity was observed. Purification of LTP was accomplished by (i) KBr density gradient ultracentrifugation, (ii) size exclusion, (iii) Cu(++) affinity and (iv) ion exchange chromatographies. LTP molecular mass was estimated approximately 770 kDa, comprising three apoproteins, apoLTP-I (315 kDa), apoLTP-II (85 kDa) and apoLTP-III (58 kDa). Phospolipid content of (32)P-LTP was determined after two-dimensional TLC. (32)P-phospholipid-labeled and unlabeled lipophorins, purified from R. prolixus were incubated in the presence of LTP resulting in the time-dependent transfer of phospholipids. LTP-mediated phospholipid transfer was not a selective process.     

Decrease in DEET repellency caused by nitric oxide in Rhodnius prolixus

N,N-diethyl-3-methylbenzamide (DEET) is widely used as an insect repellent; however, little is known about its mode of action. On the other hand, nitric oxide (NO) participates in the olfaction transduction pathway of insects. In this work, nitroso-acetyl-cysteine (SNAC), a nitric oxide donor, or dibutyril-cyclic-GMP (db-cGMP), the cyclic nucleotide analog, were applied on fifth instar nymphs of Rhodnius prolixus before exposing them to DEET, to obtain information about the possible role of NO/cGMP system in the olfaction process. In the first place, we exposed the nymphs to several DEET concentrations (70, 700, 1,750, and 3,500 microg/cm2). All these concentrations produced a repellent effect. A decrease in repellency during the course of the experiment was observed when the nymphs were exposed to high concentrations of DEET (700 and 1,750 microg/cm2), suggesting an adaptation phenomenon. The pre-treatment of the insects with 15 microg /insect of SNAC or 2 microg/insect of db-cGMP produced a reduction of the repellency. An increase in locomotor activity was observed in insects exposed to 350 or 700 microg/cm2 DEET. Although exposure to 70 microg/cm2 DEET produced a high repellency response, it did not modify the insects' locomotor activity. Insects treated with two doses of SNAC before being exposed to 350 microg/cm2 of DEET showed no differences in locomotor activity compared to controls.     

The role of salivary nitrophorins in the ingestion of blood by the triatomine bug Rhodnius prolixus (Reduviidae: Triatominae)

To assist haematophagy, Rhodnius prolixus produces several bioactive molecules in its saliva which it injects into the host skin. The most abundant of these molecules are the nitrophorins (NPs). In this work, we reduced the expression of NP1-4 in the saliva of R. prolixus by RNAi and evaluated the subsequent feeding performance of the bugs using the cibarial pump electromyogram either on the dorsal skin or on the tail vein of the mice. NPs salivary mRNA was reduced by >99% in comparison to controls. Saliva from knockdown nymphs also presented 82% less haemproteins while the total protein was not reduced. Knockdown nymphs feeding on the skin had lower ingestion rates mainly due to the longer cumulative probing time and lower cibarial pump frequency. Another difference was that knockdown insects bit approximately 5 times more. No differences were observed between groups fed on the tail vein. When the feeding sites were compared, nymphs fed on the tail vein had higher effective ingestion rates. These findings endorse the importance of the NPs for the ability of bugs to complete the meal in a short total contact time with a low number of bites, decreasing the perception of the insect by the host.     

Preliminary characterisation of digestive proteases of the green mirid, Creontiades dilutus (Hemiptera: Miridae)

Protease activities in the secreted saliva, salivary glands and midgut of the green mirid, Creontiades dilutus, were investigated. The saliva and salivary glands had more protease activity than the midgut, but no differences in protease activity levels were detected between male and female mirids, adult mirids and third instar nymphs, or between fed and starved mirids.In the salivary glands, chymotrypsin-like serine proteases predominated, as characterised by inhibitor specificity, basic pH optima, and hydrolysis of N-benzoyl-L-tyrosine p-nitroanilide and N-succinyl-ala-ala-pro-leu p-nitroanilide. The pH optimum of midgut extracts was acidic (pH 4), implying that acidic proteases predominate. However, protease activity was inhibited substantially by both aprotinin and E-64, suggesting the presence of both serine and cysteine proteases in the midgut of the green mirid.     

Identification and distribution of carbohydrate moieties on the salivary glands of Rhodnius prolixus and their possible involvement in attachment/invasion by Trypanosoma rangeli

In the present study, FITC-labelled lectins (WGA, Con A, PNA, HPA, and TPA) were utilized to investigate carbohydrate residues on the surface of Rhodnius prolixus salivary glands. The results revealed that the salivary glands are rich in carbohydrate moieties and the diversity in binding pattern of particular lectins showed the presence of specific carbohydrate residues in the basal lamina, muscle, and cell layers of the glands. Subsequently, the sugars detected on the salivary gland surface were employed to investigate the interaction between Trypanosoma rangeli and the R. prolixus salivary glands. In vitro adhesion inhibition assays using long epimastigote forms (the invasion/adhesion forms) showed that some sugars tested were able to block the receptors on both the surfaces of the salivary glands and on T. rangeli. Among the sugars tested, GlcNAc, GalNAc, and galactose showed the highest overall inhibitory effect, following pre-incubation of either the salivary glands or parasites. These results are discussed in relation to previous work on the role of carbohydrates and lectins in insect vector/parasite interactions.     

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

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

Feeding and defecation of Rhodnius (hemiptera: Reduviidae) fed human blood

Feeding and defecation behavior of Rhodnius prolixus Stal, 1859, R. robustus Larrousse, 1927, R. neivai Lent, 1953 and R. pictipes Stal, 1872, artificially fed on human blood, were studied under laboratory conditions. In all species, first instar nymphs did not defecate in the first 30 minutes after feeding. R. pictipes did not accept artificial feeding but fed directly on humans. Nymph and adult R. prolixus had a higher defecation index (DI) than other species; third instar nymphs had the highest DI = 1.62. In all instars, most individuals accepted the food in 3 Pounds minutes and finished feeding in less than 15 minutes.     

Role of phospholipids in the lipophorin particles of Rhodnius prolixus.

The lipophorin of Rhodnius prolixus metabolically labelled with 32P exclusively in the phospholipid moiety was purified on a potassium bromide gradient and treated with phospholipase A2 in the presence of an excess of fatty acid-free albumin. The treatment completely removed the phospholipids from the particles and generated [32P]-lysophosphatidylcholine, [32P]-lysophosphatidylethanolamine, and free fatty acids that remained bound to albumin. The phospholipid-depleted lipophorin particles remained soluble, indicating that phospholipids are not essential in maintaining the stability of the particles in aqueous solution. Complete removal of phospholipids did not affect the association of apolipophorin III with lipophorin particles. Lipophorin density increased slightly from 1.120 to 1.134 g/ml after treatment. The phospholipid-depleted particles also retained their ability to be recognized and loaded in vitro with phospholipids delivered by the fat body, thus supporting the concept of lipophorin's role as a reusable lipid shuttle for phospholipids.     

Proteolytic activation of canatoxin, a plant toxic protein, by insect cathepsin-like enzymes

Canatoxin is a protein isolated from jackbean (Canavalia ensiformis), seeds. Injected intraperitoneally, the toxin is lethal to mice but it is inactive if given orally. Canatoxin is also lethal when fed to insects with cathepsin-based digestion while insects with trypsin-based digestion are not affected. The hypothesis that canatoxin is proteolytically activated by cathepsins was investigated. Experiments were performed with 4(th) instar and adult Rhodnius prolixus fed meals containing canatoxin (2.5 microg/mg weight body). While 100% of nymphs died, no effect was observed in adults. Hemolymph taken from nymphs and adults showed the presence of canatoxin's proteolytic fragments. Reduced lethality was seen in R. prolixus 4(th) instars fed meals containing canatoxin and inhibitors of cathepsin enzymes, E-64 (2.0 microM) or Pepstatin-A (2. 0 microM). In another approach, canatoxin was digested in vitro with enzymes from the bruchid, Callosobruchus maculatus, and the resulting peptides were tested in R. prolixus. Three groups of toxic peptides (8,000-12,000 kD range) were separated by gel-filtration. When these peptides were fed to the insects simultaneously with the cathepsin inhibitors, no protective effect was seen. These results confirm the proteolytic activation of canatoxin by insect cathepsin-like enzymes to produce entomotoxic peptide(s). Furthermore, our data point towards overlooked differences in the digestive physiology of distinct life stages of R. prolixus. Arch.     

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

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

Differential susceptibility of triatomines of the genus Rhodnius to Trypanosoma rangeli strains from different geographical origins

The susceptibility of four Rhodnius species to different Trypanosoma rangeli strains was evaluated using both intracoelomic inoculation and oral infection. Rhodnius prolixus, Rhodnius domesticus, Rhodnius neglectus and Rhodnius nasutus were infected with Trypanosoma rangeli Macias (Venezuela), Choachi (Colombia) and SC-58 (Brazil) strains, revealing distinct haemolymph and salivary glands infection rates. The obtained infection rates were revealed to be dependent on the method of infection and the triatomine species. Our results suggest the existence of a high adaptation between the strain and the local vector.     

Defecation index and reproductive success of Triatoma maculata (Hemiptera: Reduviidae) under laboratory conditions

The reproductive and defecating behavior of Triatoma maculata (Erichson 1848) was studied on animals from an university culture in Venezuela. This species does not reach the importance of Rhodnius prolixus Stal 1859 as Chagas disease vector in Venezuela. This study addressed the role of defecating frequency, an index of how dangerous the animals are for the human population, and its relationship with why T. maculata is a less important vector than R. prolixus. Human blood was fed to the insects through an artificial feeding device. The 2nd and 3rd instar nymphs defecated more frequently (Id= 0.6, n=40), and our Vth instar nymphs did not complete sexual differentiation. Fertility was 55% (n=865) and fecundity 8 eggs/female/week (n=26). Egg incubation lasted 22 days (n=477). Female longevity was 51 days (n=26). Intermould time grew progressively from 35 days for 1st to 40 days for 4th instar nymphs (n=40). Mould percentage varied from 0% for Vth to 63% for 3rd instar nymphs. Mortality varied from 8% for 3rd to 100% for Vth instar nymphs. These results support evidences explaining the lesser vectorial capacity and low density of T. maculata in human domiciles, including reduced reproduction and defecation when the animal feeds on human blood.     

Functional proteomics of neuropeptidome dynamics during the feeding process of Rhodnius prolixus

In hematophagous insects, blood intake triggers a prompt response mediated by neuropeptides, which regulates a variety of physiological processes. Here we report a quantitative proteomic analysis of the postfeeding response in the central nervous system of Rhodnius prolixus, a vector of Chagas disease. The concentration of neuropeptides NVP-like, ITG-like, kinin-precursor peptide, and neuropeptide-like precursor 1 (NPLP1) significantly changes in response to blood intake. We also performed a neuropeptidomic analysis of other feeding-related organs, namely salivary glands and gut. We identified NPLP1 in salivary glands and myosuppressin in midgut. This is the first report suggesting a role for NPLP1, involving the peptides processed from this precursor in the hormonal control of the production and/or release of saliva. Our results contribute to the understanding of the postprandial neuroendocrine response in hematophagous and provide important information for physiological and pharmacological studies aimed to the design of next-generation insecticides such as peptidomimetics.     

Exploring the sialome of the blood-sucking bug Rhodnius prolixus

Rhodnius prolixus is a Hemiptera that feeds exclusively on vertebrate blood in all life stages. Its salivary glands produce potent pharmacological substances that counteract host hemostasis, including anti-clotting, anti-platelet, and vasodilatory substances. To obtain a further insight into the salivary biochemical and pharmacological complexity of this insect, a cDNA library was randomly sequenced, and salivary gland homogenates were fractionated by HPLC to obtain aminoterminal sequences of abundantly expressed proteins. Results indicate a remarkable expansion of the lipocalin family in Rhodnius salivary glands, among other protein sequences described. A summary of 31 new full length proteins deducted from their mRNA sequence is described, including several new members of the nitrophorin, triabin, and pallidipin families. The electronic version of the complete tables is available at http://www.ncbi.nlm.nih.gov/projects/vectors/rhodnius_prolixus.     

Serotonin: a coordinator of feeding-related physiological events in the blood-gorging bug, Rhodnius prolixus

Rhodnius prolixus is an obligatory blood-feeder that can ingest blood meals of up to 10 times its mass. Rapid production of urine commences within 2-3 min of the start of feeding in order to eliminate the load of water and salts, and so there is an increase of Malpighian tubule secretion greater than 1,000 fold in response to feeding. Feeding and post-prandial diuresis in Rhodnius are highly coordinated events, including for example, host recognition, probing, injection of saliva, cuticle plasticization, passage of blood through the digestive system, diuresis and excretion. This review illustrates that many of the known functions of serotonin in Rhodnius are feeding-related. Serotonin coordinates or 'orchestrates' feeding-related physiological events either as a neurotransmitter/neuromodulator, delivered to target tissues in the nerve supply, or as a neurohormone, delivered by the haemolymph. Thus, serotonin has physiological effects upon the salivary glands, cuticle, digestive tract, cardiac muscle, and Malpighian tubules. By discussing these aspects, the review illustrates that serotonin acts in a coordinated manner to prepare Rhodnius for this energy-demanding process of feeding and diuresis.