Rhodnius heme-binding protein (RHBP) is a heme source for embryonic development in the blood-sucking bug Rhodnius prolixus (Hemiptera, Reduviidae)

We have previously shown that Rhodnius prolixus' eggs and hemolymph are pink due to the presence of the hemeprotein Rhodnius heme-binding protein (RHBP). In the hemolymph it functions as an antioxidant. Nevertheless, its function in eggs has not been determined. Here we present evidence that RHBP is a source of heme for embryonic development. RHBP content decreases during embryogenesis, but the total heme content of eggs remains unchanged. Biliverdin, the product of heme degradation, is not detectable in late embryos. The activity of the heme-synthesizing pathway is low throughout embryogenesis and rises sharply after nymphs' hatching. Heme-radiolabeled eggs were produced and, at the day of hatching, nymphs were dissected. The presence of radiolabeled heme in their carcass is an indication that heme reutilization is occurring. The only animal known to reutilize heme in significant levels is the cattle tick Boophilus microplus, which cannot synthesize its own heme. Diversely, Rhodnius can synthesize its own heme but, in the context of embryogenesis, heme demand seems to be supplied by the programmed release of heme form RHBP. This behavior indicates that in Rhodnius, we might have a highly unusual profile: heme is both synthesized and reutilized.     

Antidiuretic mechanism in Rhodnius prolixus (Stal, 1859) (Hemiptera, Reduviidae)

An antidiuretic mechanism is proposed for Rhodnius prolixus, whose activity is manifested when the diuretic phase has terminated and is maintained until the insect has fed again. Presumably this mechanism acts at the level of the proximal rectal sphincter and is inhibited by ingestion, mechanical distension of the gut and central disinhibition by decapitation. It is suggested that the antidiuretic activity is maintained by the nervous system and is modified when there is distention of the gut or when the nervous signal is interrupted by decapitation. It is demonstrated that the excretion of urine in R. prolixus is not controlled exclusively by the diuretic hormone, but rather that factors not linked to the haemolymph maintain the insects in a diuretic or nondiuretic state.     

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.     

Lipid metabolism in Rhodnius prolixus (Hemiptera: Reduviidae): role of a midgut triacylglycerol-lipase

The utilization of dietary lipids was studied in adult females of Rhodnius prolixus with the use of radiolabeled triacylglycerol (TAG). It was shown that (3)H-triolein, when added to blood meal, was hydrolyzed to free fatty acids in the posterior midgut lumen. Subsequently, free fatty acids were absorbed by posterior midgut epithelium and used in the synthesis of phospholipids, diacylglycerol (DAG) and TAG. Phospholipids, DAG and free fatty acids were then found in hemolymph, from where they were rapidly cleared, and label was found in the fat body, mainly associated with TAG. Radioactive lipids, especially TAG and phospholipids, also accumulated in the ovaries. The TAG-lipase activities of posterior midgut luminal content and tissue were characterized by incubation of these samples with (3)H-triolein in the presence of the detergent Triton X-100 and determination of the amounts of released radioactive free fatty acids. Under the conditions employed here, the release of free fatty acids was proportional to the incubation time and to the amount of sample obtained from insect midgut (enzyme source) that was added. TAG-lipase activities were affected by pH and posterior midgut tissue showed optimum activity around pH 7.0-7.5, but the luminal content had the highest activities as pH decreased. Differences in activities were observed according to calcium concentration in the medium. TAG-lipase activities were also affected by the concentration of NaCl and were activated in the presence of increasing salt concentrations. These activities were inhibited by phenylmethylsulphonyl fluoride (PMSF). On the second day after blood meal, when digestion is very intense, TAG-lipase activities were maximal and then gradually decreased.     

Fine structure of antennal mechanosensilla of adult Rhodnius prolixus stål (Hemiptera: Reduviidae)

Each antenna of both sexes of adult Rhodnius prolixus has approximately 570 mechanosensitive neurons that innervate five morphologic types of cuticular mechanosensilla: campaniform sensilla, tapered hairs, trichobothria, and type I and type II bristle sensilla. Each campaniform sensillum and tapered hair is presumably innervated by one mechanosensitive bipolar neuron and probably functions in proprioception. The campaniform sensilla being located at the base of the scape could monitor the position of the antenna. Tapered hairs are found at the distal margin of flagellar segment I and projecting laterally from the bases of the pedicel and scape. They probably provide information about the relative positions of the antennal segments. Seven trichobothrium are located on the pedicel and three on flagellar segment I. Each trichobothrium has a long filamentous hair inserted into the base of a socket that extends inwardly as a cuticular tube and is innervated by one bipolar neuron with a tublar body, a parallel arrangement of microtubules associated with electron-dense material. The trichobothria may respond to small variations in air currents. Type I bristles occur at the base of the antenna and are the most numerous type of mechanosensillum; an average of 452 occur on each antenna of females and 440 on males. The bristle is curved toward the antennal shaft and is serrated distally. Type II bristles are located distally and are the second most numerous type of mechanosensillum; an average of 88 were counted on each antenna of females and 94 on males. The type II bristle is straight with small, longitudinal, external grooves and projects laterally from the antennal shaft. Each type I and II bristle sensillum is innervated by a bipolar neuron whose dendrite is divided into an inner and outer segment. The outer segment is encased by a dendritic sheath which may be highly convoluted and distally contains a tubular body. Two sheath cells are associated with each sensillum. Both types of bristle sensilla have a tactile function. The tubular bodies of both types of bristle sensilla have a complex structure indicating that they are very sensitive. Variations in the amount and arrangement of the electron-dense material at the tip of the tubular bodies may reflect differences in viscoelastic properties that underlie functional characteristics.     

Morphometric analysis of Rhodnius prolixus Stal (Hemiptera:Reduviidae) midgut cells during blood digestion

Post-feeding ultrastructural modifications to the midgut cells of Rhodnius prolixus are quantified using morphometry. Changes in relative and absolute volumes and/or surface areas are demonstrated for the whole cells, nuclei, mitochondria, rough endoplasmic reticulum, lysosomes, Golgi apparatus, storage vesicles, glycogen, microvilli, and basal labyrinth, before and during blood digestion. These parameters are separately determined for cells from each of the three midgut regions, and are correlated against previously published cycles of digestive enzyme activities. The results support the proposed division of the midgut of R. prolixus into three functional regions: the anterior midgut or crop is the site of water transport immediately after feeding, and of lipid and glycogen storage. No protein digestion occurs in this region. The anterior intestine is the site of most proteinase synthesis and secretion, although limited absorption and nutrient storage also occurs. The posterior intestine is responsible for some secretory activity, but is also implicated as the most important region for absorption of digested nutrients and for carbohydrate absorption and storage.     

Effects of juvenile hormone analogue on ecdysis prevention induced by precocene in Rhodnius prolixus (Hemiptera:Reduviidae)

Precocene II, added to the meal of fourth-instar larvae of Rhodnius prolixus (25 micrograms/ml of blood), induced an increase in the duration of the molting cycle. This effect was related to the decrease of both the nuclear area of the prothoracic gland cells and the mitotic activity in epidermal cells. Juvenile hormone analogue applied topically (60 micrograms/insect) together with Precocene II treatment avoided atrophy of the prothoracic glands and induced a higher number of epidermal mitosis accelerating the time of subsequent ecdysis. A possible relationship between juvenile hormone and production of ecdysone is discussed.     

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.     

The effects of artificial diets on the anterior intestinal cell ultrastructure of Rhodnius prolixus (Hemiptera:Reduviidae)

Mated female Rhodnius prolixus were fed diets of washed rabbit erythrocytes (RBC), rabbit plasma, edestin (a plant storage protein) in Ringer's or Ringer's solution alone. All diets contained 1 mM ATP. The effects of these diets on anterior intestinal cell ultrastructure were evaluated and compared to changes induced by normal blood feeding. Separation of basal labyrinth membranes was induced earlier after Ringer's-feeding than with all other diets. Normal modifications of the rough endoplasmic reticulum (rer) were observed only in response to blood components, while rer cisternae were swollen shortly after edestin feeding. Normal modifications to lysosomal structure were induced by plasma and RBC feeding, but changes were also observed with the non-blood diets. Production of extracellular membrane layers (ECML) by the microvilli occurred in response to each diet: plasma-feeding induced considerably early ECML formation, but prolonged ECML proliferation was greatest after RBC feeding. Storage inclusions were most prominent in cells of plasma-fed insects. Initial changes in the basal labyrinth may be induced by a high luminal fluid: protein ratio; in the rer by blood factor(s); and in lysosomes by plasma factor(s). Gut distention initiates ECML production but continued proliferation is regulated by the midgut contents.     

Fine structure of antennal putative thermo-/hygrosensilla of adult Rhodnius prolixus Stål (Hemiptera: Reduviidae)

At least five nonporous sensilla with inflexible sockets (npsensilla) occur on each antenna of both sexes of adult Rhodnius prolixus. Externally the sensillum appears as a short, rounded peg set into a pit surrounded by a depression. A very electron-dense material occurs in the peg lumen and the inner aspect of the pit. Filamentous extensions of this material radiate into the overlying outlets. Each sensillum is innervated by three neurons with unbranched dendrites. Two dendrites extend to the peg tip and distally are covered by a dendritic sheath. The portion of these dendrites within the sheath contains a large number of microtubules. The third dendrite terminates near the base of the dentritic sheath and partially wraps around the other two dendrites. Three sheath cells are associated with each sensillum. Based on similarities in structure with sensilla of known function it is probable that the np-sensilla of R. prolixus are thermo-/hygrosensilla responding to cold, dryness and wetness. The sensilla have a number of structural similarities with insect rectal sheath cells known to absorb atmospheric water by electroosmosis. Possibly this process leads to volumetric alterations of cuticular elements associated with the dendrites and ultimately to mechanotransduction.     

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.     

Biological effect of 1-dodecanol in teneral and post-teneral Rhodnius prolixus and Triatoma infestans (Hemiptera: Reduviidae)

Topical application of 1-dodecanol was significantly more toxic against teneral first nymphs (1-3 h old) than post-teneral first nymphs (24 h old). The lethal dose ratios were 711,500 for Rhodnius prolixus and 3613 for Triatoma infestans. No significative difference between LD50 was found when 1-dodecanol was injected in recently hatched adult R. prolixus (1-4 h old) nor in older adults (24 h old). These values were similar to those calculated for deltamethrin (an effective triatomicide), showing that 1-dodecanol had no insecticidal properties when it was applied by injection. Topical application of high dose of 1-dodecanol (1 microg/i) on teneral first nymphs of R. prolixus, produced an interruption of the darkening process of the cuticle, and probably in the development of its physiological properties.     

Development of Rhodnius prolixus (Hemiptera: Reduviidae) under constant and cyclic conditions of temperature and humidity.

Development of Rhodnius prolixus after eclosion until the adult stage was studied at constant temperatures (T), 15, 20, 25, 28, 35 C, and relative humidities (RH), 75, 86 and 97%, and fluctuating (16/8 hr) temperatures, T I/II, 15/28 C, 20/25 C, 25/28 C and 25/35 C, and relative humidities, RH I/II, 86/75% and 97/75%. Eclosion or molting were not observed at 15 C and 86 or 97% RH, respectively. At 35 C and 75% RH only few insects molted. By alternating T I/II, 15/28 C and 25/35 C, insects developed at high frequency. Cumulating the average lengths of the interphases within independent groups for each instar, R. prolixus reached the adult stage most rapidly (86.7 days) and at highest frequency per instar (mean: 91.8%) at 28 C and 75% RH. Under fluctuating T I/II, development was completed within 100 days or less at 25/28 C and 25/35 C with high rates of hatch and molting. Development was slowest at fluctuating TI/II, 15/28 C and 20/25 C (>185 days), and at constant 20 C (>300 days). Mortality was higher at constant 97% RH or fluctuating RH I, 97%, than at constant or fluctuating 86% RH. Refeeding was minimal at optimal conditions of T and RH for development. The most refeeding was observed at a constant 35 C.     

Effect of human blood feeding on the fecundity, fertility and biological cycle of Rhodnius prolixus (Hemiptera: Reduviidae)

The effect of several human blood fractions artificially fed to Rhodnius prolixus Stal 1859 on oviposture (fecundity), egg-hatching (fertility) and life cycle was observed. Specimens fed on man's blood were more fecund than those fed with woman's blood. There were no significant differences in fertility related to host sex. The nymphal development time and number of feedings to molt to the following instar were estimated. Animals fed only on blood plasm did not finish nymphal development, while those fed only blood red cells ended their life cycle in the third nymphal instar. Total life cycle lasts 129 days in individuals fed with whole blood.     

Changes associated with laboratory rearing in antennal sensilla patterns of Triatoma infestans, Rhodnius prolixus, and Rhodnius pallescens (Hemiptera, Reduviidae, Triatominae)

We examined changes in the array of antennal sensilla of three species of Triatominae (Triatoma infestans, Rhodnius prolixus, and R. pallescens) following their establishment for different periods in laboratory culture. In each case, the laboratory colonies were compared with conspecific samples taken directly from the field, by quantitative analysis of the sensilla arrays on the three distal segments of the antenna in terms of the densities of three types of chemoreceptors (basiconics and thick and thin walled trichoids) and one type of mechanoreceptor (bristles). Sensilla densities were compared by ANOVA or non-parametric tests, and by multivariate discriminant analysis. Strains of the same species reared in different laboratories showed significant differences in their sensilla arrays, especially when compared to field-collected material from the same geographic origin. A Bolivian strain of T. infestans reared in the laboratory for 15 years and fed at monthly intervals, showed greatest differences from its conspecific wild forms, especially in terms of reductions in the number of chemoreceptors. By contrast, an Argentine strain of T. infestans reared for 25 years in the laboratory and fed weekly, showed a relative increase in the density of mechanoreceptors. A Colombian strain of R. prolixus reared for 20 years and fed weekly or fortnightly, showed only modest differences in the sensilla array when compared to its wild populations from the same area. However, a Colombian strain of R. pallescens reared for 12 years and fed fortnightly, did show highly significant reductions in one form of chemoreceptor compared to its conspecific wild populations. For all populations, multivariate analysis clearly discriminated between laboratory and field collected specimens, suggesting that artificial rearing can lead to modifications in the sensory array. This not only supports the idea of morphological plasticity in these species, but also suggests caution in the use of long-established laboratory material for experimental studies designed to extrapolate the natural behaviour and physiology of these species.     

Sulfated glycosaminoglycans in two hematophagous arthropod vectors of Chagas disease, Triatoma brasiliensis and Rhodnius prolixus (Hemiptera: Reduviidae)

The characterization of sulfated glycosaminoglycans (GAGs) in hematophagous arthropod vectors in general has been limited, with the exception of the studies in the triatomine Rhodnius prolixus. Heparan sulfate (HS) and chondroitin sulfate (CS) were previously identified and structurally characterized in extracts of whole bodies of fourth instar larvae of R. prolixus. Recently, we showed the expression of these two sulfated GAGs in specific body tissues of adult males and females and in embryos of R. prolixus. In the present work, we identified and compared the sulfated GAG composition in specific tissues of adult insects and in embryos of another triatomine species, Triatoma brasiliensis. Sulfated GAGs were isolated from the fat body, intestinal tract, and the reproductive tracts of adult insects and from embryos. Only HS and CS were found in the tissues analyzed. The present results extend the initial observations on the sulfated GAG composition in R. prolixus by showing that these molecules are widely distributed among internal organs of triatomines. These observations may be useful for future investigations aiming to evaluate the possible implication of these compounds in physiological events that take place in a specific organ(s) in these insects.     

Scalariform junctions in the malpighian tubules of the insects Rhodnius prolixus (Hemiptera: Reduviidae) and Aedes taeniorhynchus (Diptera: Culicidae)

The ultrastructure of scalariform junctions in the Malpighian tubules of the hemipteran Rhodnius prolixus and the dipteran Aedes taeniorhynchus is described. Both autocellular and intercellular scalariform junctions are illustrated. This is the first report of scalariform junctions in the Malpighian tubules of a dipteran. When combined with previous observations by other authors, the presence of scalariform junctions has now been reported in the Malpighian tubules of insects from five orders, including ametabolous, hemimetabolous, and holometabolous forms. The cell types in which scalariform junctions were found in R. prolixus and A. taeniorhynchus differ in the direction of ion and fluid transport. The cells share the capacity to transport KCl. These same cells also possess morphological features promoting close associations of mitochondria and plasma membranes in the apical region of the cell. The possible role of scalariform junctions is discussed in light of these observations.     

Prolixase, a fibrinolytic enzyme isolated from Rhodnius prolixus (Heteroptera, Reduviidae): antigenic properties

Antigenic properties of prolixase are studied showing that this fibrinolytic enzyme, induces in the rabbit, the formation of precipitating but slightly, hemagglutinating homologous antibodies.