Salivation pattern of Rhodnius prolixus (Reduviidae; Triatominae) in mouse skin

The objective of this work was to study the pattern of salivation of triatomines during feeding in mouse skin. Rhodnius prolixus was fed with a solution of the dye acridine orange or fluorescein. The saliva was efficiently labelled with acridine orange, probably due to the difference in pH between the salivary gland (6.0) and the hemolymph (6.5-7.0). This procedure was not effective at labelling the saliva of Triatoma infestans, however, fluorescent labelling of R. prolixus saliva allowed us to demonstrate that salivation occurs during entire feeding process. The saliva is released soon after the bite. In the probing phase, saliva is pumped continuously in the host skin, including around the blood vessels. During the engorgement phase, saliva is observed in a bolus within the blood vessel and some of it is sucked up by the insect, together with blood. The frequency of saliva emission inside the vessels was low (0.51+/-0.18 Hz). The saliva deposition in the microcirculation is continuous and modulated by the frequency of the cibarial pump because, when functioning at high frequency, cibarial pump sucks almost all saliva to the insect gut. This mechanism would determine the quantity of saliva deposited in the microcirculation as necessary, and consequently minimizing the host's immune response to salivary antigens.     

Trypanosoma cruzi: Multiplex PCR to detect and classify strains according to groups I and II

A multiplex PCR was developed for simultaneous detection of Trypanosoma cruzi DNA and classification of the parasite strain into groups I and II. As little as 10 fg of T. cruzi DNA could be detected by multiplex PCR. The technique was shown to be specific for T. cruzi DNA, since no PCR amplification products were obtained with DNA from other tripanosomatid species. Multiplex PCR was validated by assaying genomic DNA from 34 strains of T. cruzi that had been previously characterized; 24 blood samples from experimentally-infected mice and non-infected controls; 20 buffy coat samples from patients in the acute phase of Chagas disease and non-infected individuals, and 15 samples of feces from naturally-infected Triatoma infestans. T. cruzi samples from patients and from Y strain-infected mice were classified by multiplex PCR as T. cruzi II and samples from T. infestans and Colombiana strain-infected mice as T. cruzi I.     

Interactome: Smart hematophagous triatomine salivary gland molecules counteract human hemostasis during meal acquisition

Human populations are constantly plagued by hematophagous insects' bites, in particular the triatomine insects that are vectors of the Trypanosoma cruzi agent in Chagas disease. The pharmacologically-active molecules present in the salivary glands of hematophagous insects are injected into the human skin to initiate acquisition of blood meals. Sets of vasodilators, anti-platelet aggregators, anti-coagulants, immunogenic polypeptides, anesthetics, odorants, antibiotics, and detoxifying molecules have been disclosed with the aid of proteomics and recombinant cDNA techniques. These molecules can provide insights about the insect-pathogen-host interactions essential for understanding the physiopathology of the insect bite. The data and information presented in this review aim for the development of new drugs to prevent insect bites and the insect-transmitted endemic of Chagas disease.     

Amines from vertebrates guide triatomine bugs to resources

Most triatomine bugs (Heteroptera: Reduviidae) are nest-living insects that require vertebrate blood or invertebrate haemolymph to complete their life cycle. Vertebrates accumulate excretory products in or near their nesting sites and we hypothesize that triatomines use emanations from such host wastes when searching for resources. Here we recount how triatomine bugs increase upwind locomotion on a servosphere in response to volatile amine constituents of vertebrate excretions. Fresh chicken faeces is strongly attractive to Rhodnius prolixus nymphs. Ammonia induces attraction and an increase in both speed and total path length by R. prolixus on the servosphere. Whereas ethylamine and dimethylamine attract R. prolixus, Triatoma infestans and Panstrongylus geniculatus, other amine constituents of vertebrate excretions such as isobutylamine and hexylamine induce R. prolixus nymphs to walk faster and for a longer period. These amines are derived from generally occurring metabolites of vertebrates and from gut flora metabolism. We conclude that amines and other products associated with nesting hosts serve as signals for foraging triatomines.     

Appetence behaviours of the triatomine bug <Emphasis Type="Italic">Rhodnius prolixus</Emphasis> on a servosphere in response to the host metabolites carbon dioxide and ammonia

A combination of 1,000 ppm CO(2) plus 30-40 ppb NH(3) in an air stream induced Rhodnius prolixus nymphs walking on a servosphere to perform a series of appetence behaviours. Shortly after the onset of stimulation the nymphs turned sharply upwind towards the source of the chemostimuli (within 13 +/- 9 s) from mostly downwind and crosswind walks in the air stream alone. The mean vector angles of these upwind tracks were concentrated in a cone 60 degrees either side of due upwind. The upwind walking bugs stopped more frequently but for a shorter duration and walked at a higher speed than before stimulation. During stops in the presence of the chemostimuli the bugs frequently corrected their course angles and extended their forelegs to reach higher with their antennae in the air. In the air stream alone, R. prolixus nymphs frequently sampled the sphere surface with the antennae and cleaned their antennae with the foreleg tarsi. However, the nymphs only briefly tapped the left or right antennal flagellum on the corresponding first leg tarsus and never touched the servosphere surface in the presence of the chemostimuli. After chemostimulus removal from the air stream the bugs continued to respond with the same appetence responses as during stimulation, but walked more tortuously in a crosswind direction in an effort to regain contact with the chemostimuli.     

Influence of the intestinal anticoagulant in the feeding performance of triatomine bugs (Hemiptera; Reduviidae)

Triatomines are haematophagous insects in all post-embryonic life stages. They are vectors of Trypanosoma cruzi, the causative agent of Chagas disease. Their vectorial ability is influenced by their feeding performance, which varies greatly amongst species. Recent work showed that inhibition of the coagulation process in the anterior midgut (crop) environment considerably influences the blood meal size. In this work, we performed a comparative study of the level of anticoagulant activity in the saliva and crop contents of three triatomine species - Triatoma infestans, Triatoma brasiliensis and Rhodnius prolixus - and correlated this with their feeding performance on live hosts. Moreover, the feeding parameters on a large diameter vessel influenced by the crop anticoagulants were evaluated in detail. The anticoagulant activity was significantly higher in the crop contents than in salivary glands, varying from 1.6-fold higher for R. prolixus to 70-fold higher for T. brasiliensis. Amongst the species, T. brasiliensis had the lowest crop anticoagulant activity, the lowest concentration of thrombin inhibitor, and took the longest to feed. Triatoma brasiliensis nymphs that had their intestinal anticoagulant (brasiliensin) knocked down by RNA interference had the lowest capacity to maintain cibarial pump frequency at higher levels throughout the feeding process and consequently a lower ingestion rate (mg/min), even when fed under favourable conditions (large diameter vessel). However, the feeding difficulty for brasiliensin knockdown T. brasiliensis nymphs was reversed by treating the host mice with heparin (a potent systemic anticoagulant) before blood feeding. The results indicate that crop anticoagulant activity influences modulation of the blood-pumping frequency to the intestine and significantly affects the feeding efficiency of triatomine spp. on live hosts.     

Brasiliensin: A novel intestinal thrombin inhibitor from Triatoma brasiliensis (Hemiptera: Reduviidae) with an important role in blood intake

Every hematophagous invertebrate studied to date produces at least one inhibitor of coagulation. Among these, thrombin inhibitors have most frequently been isolated. In order to study the thrombin inhibitor from Triatoma brasiliensis and its biological significance for the bug, we sequenced the corresponding gene and evaluated its biological function. The T. brasiliensis intestinal thrombin inhibitor, termed brasiliensin, was sequenced and primers were designed to synthesize double strand RNA (dsRNA). Gene knockdown (RNAi) was induced by two injections of 15mug of dsRNA into fourth instar nymphs. Forty-eight hours after the second injection, bugs from each group were allowed to feed on hamsters. PCR results showed that injections of dsRNA reduced brasiliensin expression in the anterior midgut by approximately 71% in knockdown nymphs when compared with controls. The reduction in gene expression was confirmed by the thrombin inhibitory activity assay and the citrated plasma coagulation time assay which showed activity reductions of approximately 18- and approximately 3.5-fold, respectively. Knockdown nymphs ingested approximately 39% less blood than controls. In order to confirm the importance of brasiliensin in blood ingestion, fourth instar nymphs were allowed to ingest feeding solution alone or feeding solution containing 15U of thrombin prior to blood feeding. Fifty-five percent less blood was ingested by nymphs which were fed thrombin prior to blood feeding. The results suggest that anticoagulant activity in the midgut is an important determinant of the amount of blood taken from the host. The role of anticoagulants during blood ingestion is discussed in the light of this novel insight.     

Ultrastructure and receptor cell responses of the antennal grooved peg sensilla of Triatoma infestans (Hemiptera: Reduviidae)

Ultrastructural examination of grooved-peg (GP) sensilla on the antenna of fifth instar Triatoma infestans nymphs by scanning electron microscopy and transmission electron microscopy reveal that they are 8–18 μm long with a diameter of about 2–2.8 μm at the non-articulated base. Some pegs have a terminal pore. These double-walled wall-pore (dw-wp) sensilla have an outer cuticular wall with 13–18 longitudinal grooves at the distal part of the peg. Groove channels are present at the bottom of the grooves from which radial spoke channels lead into the inner sensillum-lymph cavity. A dendrite sheath connects the tip of the thecogen cell to the inner cuticular wall thus forming separated outer and inner sensillum-lymph cavities. Four or five bipolar receptor cells are ensheathed successively within the GP sensilla by the thecogen cell, trichogen and tormogen cells. The inner dendritic segments of each sensory cell give rise at the ciliary constriction to an unbranched outer dendritic segment which can reach the tip of the sensillum.Electrophysiological recordings from the GP sensilla indicate that they house NH₃, short-chain carboxylic acid and short-chain aliphatic amine receptor cells and can be divided into three functional sub-types (GP 1–3). All GP sensilla carry a receptor cell excited by aliphatic amines, such as isobutylamine, a compound associated with vertebrate odour. GP type 1 and 2 sensilla house, in addition, an NH₃-excited cell whereas the type 2 sensilla also contains a short-chain carboxylic acid receptor. No cell particularly sensitive to either NH₃ or carboxylic acids was found in the grooved-peg type 3 sensilla. GP types 1, 2 and 3 represent ca. 36, 10 and 43% of the GP sensilla, respectively, whereas the remaining 11% contain receptor cells that manifest normal spontaneous activity but do not respond to any of the afore mentioned stimuli.     

Trypanosoma cruzi: attachment to perimicrovillar membrane glycoproteins of Rhodnius prolixus

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

Hunger is the best spice: Effects of starvation in the antennal responses of the blood-sucking bug Rhodnius prolixus

Blood-sucking insects strongly rely on olfactory cues to find their vertebrate hosts. As in other insects with different lifestyles, it has been shown that endogenous and exogenous factors modulate olfactory responses. The triatomine bug Rhodnius prolixus is an important vector of Chagas disease and a classical model for studies of physiology and behavior. In this species, the behavioral response to host-derived odorants is modulated by both the time of the day and the starvation. Here I investigated the peripheral neural mechanisms underlying these modulatory effects. For this, I measured the electroantennogram (EAG) responses of insects towards different concentrations (from 0.5% to 75% vol/vol) of an attractive host-odorant, ammonia. I tested the responses of starved and fed animals during the middle of the day (when insects are inactive and aggregated in refuges) and at the beginning of the night (when insects become active and search for hosts). Regardless of the time of the day and the starvation status, EAG responses systematically increased with odorant concentration, thus accurately reflecting the response of olfactory receptor cells. Interestingly, the EAG responses of starved insects were larger than those of fed insects only during the night, with larger differences (6–7 times) observed at low–middle concentrations. This study is the first reporting modulation of sensory responses at the neural level in triatomines. This modulation, considering that triatomine hosts are mostly diurnal and are also potential predators, has an important adaptive value, ensuring that insects search for hosts only when they are hungry and at appropriate times.