The distribution of a kinin-like peptide and its co-localization with a CRF-like peptide in the blood-feeding bug, Rhodnius prolixus

Rhodnius prolixus, a blood-feeding hemipteran insect, ingests large meals which are followed by rapid diuresis to eliminate excess water and salt. In Rhodnius, serotonin and an unidentified peptide(s) [33,34] have been shown to act as neurohormonal diuretic factors. In other insects, two families of diuretic peptides, the corticotropin releasing factor (CRF)-like, and kinin peptides [9], have been identified and sequenced. Recently, we demonstrated the presence of a CRF-like diuretic peptide in the CNS and digestive system of Rhodnius [47] using immunohistochemistry and bioassay.In this study, combining immunohistochemistry and radioimmunoassay (RIA) techniques, we show the presence of leucokinin-like peptide(s) in the CNS and digestive system of Rhodnius 5th instar. Additionally, double-label immunohistochemistry demonstrates that the leucokinin-like and CRF-like peptides are co-localized in the posterior lateral neurosecretory cells of the mesothoracic ganglionic mass (MTGM) and in neurohaemal areas on abdominal nerves one and two, suggesting the possibility of co-release of the peptides into the hemolymph.Partially purified extracts of the CNS and neurohaemal tissue were tested in vitro on Malpighian tubule secretion and cAMP assays. The factors eluting with increasing acetonitrile percentages from Sep-Pak cartridges were assayed in the presence or absence of ketanserin, a serotonin antagonist which blocks the effects of serotonin on Malpighian tubules. The results indicate activity of serotonin and a CRF-like diuretic peptide on Rhodnius Malpighian tubules, but fail to demonstrate activity of the leucokinin-like peptide(s).The rapid diuresis following feeding is a highly coordinated event, requiring the movement of water and salt across the epithelial cells of the crop into the hemolymph, and from the hemolymph across the cells of the Malpighian tubules. The urine then travels along the Malpighian tubules into the hindgut in order to be expelled. The presence of a leucokinin-like peptide(s) in the CNS and digestive system, which co-localizes with a CRF-like peptide(s), suggests that kinins may play a role in the rapid diuresis, although possibly not directly on the Malpighian tubules.     

The distribution and function of serotonin in the large milkweed bug,Oncopeltus fasciatus. a comparative study with the blood-feeding bug,Rhodnius prolixus

The blood-feeding hemipteran, Rhodnius prolixus, ingests a large blood meal at the end of each larval stage. To accommodate and process this meal, its cuticle undergoes plasticisation, and its gut and Malpighian tubules respectively absorb and secrete a large volume of water and salts for rapid diuresis. Serotonin has been found to be integral to the feeding process in this animal, along with a diuretic peptide(s). The large milkweed bug, Oncopeltus fasciatus, tends to feed in a more continuous and abstemious manner, and therefore may have different physiological requirements than the blood feeder. Unlike R. prolixus, O. fasciatus is lacking serotonin-like immunoreactive dorsal unpaired median neurons in the mesothoracic ganglionic mass, and lacks serotonin-like immunoreactive neurohaemal areas and processes on the abdominal nerves, integument, salivary glands, and anterior junction of the foregut and crop. The salivary glands and crop do, however, respond to serotonin with increased levels of cAMP, while the integument and Malpighian tubules do not. In addition, O. fasciatus Malpighian tubules respond to both O. fasciatus and R. prolixus partially purified CNS extracts, which are likely to contain any native diuretic peptides. Thus, while serotonin and diuretic peptides may be involved in tubule control in R. prolixus, the latter may be of greater importance in O. fasciatus.     

Evidence for CRF-like and kinin-like peptides as neurohormones in the blood-feeding bug,Rhodnius prolixus

In Rhodnius prolixus, the rapid post-feeding diuresis is under neurohormonal control. While serotonin has been demonstrated to be a diuretic neurohormone [J Exp Biol 156 (1991) 557], a peptide is also known to be involved. Previously, we have demonstrated the presence of corticotropin releasing factor (CRF)-like and kinin-like peptides in the central nervous system (CNS) of 5th instar Rhodnius [J Exp Biol 202 (1999) 2017; Peptides 22 (2001) 161]. These peptides are present in neurohemal sites of the corpus cardiacum and are co-localized in neurohemal sites on abdominal nerves. While various CRF-like peptides have been demonstrated to increase Rhodnius Malpighian tubule secretion the kinin-like peptides do not [Peptides 23 (2002) 671]. The kinin-like peptides do however, increase hindgut contraction which may contribute to the rapid post feeding diuresis by the mixing of hemolymph and/or hindgut contents and the removal of wastes. The presence of these peptides in neurohemal sites suggests that they could be released into the hemolymph and act as neurohormones.We have used immunohistochemical techniques and radioimmunoassay (RIA) to demonstrate qualitative and quantitative changes of CRF-like and kinin-like peptides in the CNS associated with feeding. As well we have examined Malpighian tubule secretion in response to assays of hemolymph from unfed and fed insects. Hemolymph was also partially purified by Sep-Pak and HPLC and the fractions assayed for kinin-like immunoreactivity and the ability to stimulate Malpighian tubule secretion. The results suggest that both kinin-like and CRF-like peptides are neurohormones in Rhodnius, released in response to feeding.     

The biological activity of diuretic factors in Rhodnius prolixus

The rapid post-feeding diuresis of Rhodnius prolixus is under neurohormonal control and involves the integrated activity of the crop, Malpighian tubules and hindgut. One of the factors which is involved in this rapid diuresis is serotonin, however a peptide(s) is also considered to be involved. In other insects, corticotropin releasing factor (CRF)-like and kinin-like, calcitonin-like peptides and CAP(2b) have been demonstrated to be diuretic factors/hormones.In the present study, serotonin and CRF-like peptides increased secretion rate and cAMP content of Rhodnius Malpighian tubules, while the kinin-like peptides tested did not increase secretion rate or cAMP content of the tubules. Extracts of the CNS were processed and several HPLC fractions revealed kinin-like immunoreactivity but these fractions did not increase secretion rate when tested on Malpighian tubules. However, these same fractions did possess activity when tested on the hindgut contraction assay. In addition, material eluting at higher acetonitrile concentrations from the HPLC increased secretion and cAMP content of Rhodnius Malpighian tubules. This material eluted at concentrations of acetonitrile consistent with the elution time of CRF-like peptide standards.Synergism was demonstrated using the pharmacological agent forskolin and serotonin, tested on the rate of secretion of Rhodnius Malpighian tubules, in agreement with data of Maddrell et al. As well, synergism could be demonstrated using mesothoracic ganglionic mass (MTGM) homogenates and serotonin at some concentrations of serotonin. However, combinations of CRF-like material and serotonin increased secretion additively, not synergistically. Kinin-like peptides, tested along with CRF-like material and serotonin, at low concentrations, did not increase secretion above that of those factors tested alone.     

Allatotropin Modulates Myostimulatory and Cardioacceleratory Activities in Rhodnius prolixus (Stal).

Haematophagous insects can ingest large quantities of blood in a single meal and eliminate high volumes of urine in the next few hours. This rise in diuresis is possible because the excretory activity of the Malpighian tubules is facilitated by an increase in haemolymph circulation as a result of intensification of aorta contractions combined with an increase of the anterior midgut peristaltic waves. It has been previously described that haemolymph circulation during post-prandial diuresis is stimulated by the synergistic activity of allatotropin (AT) and serotonin in the kissing bug Triatoma infestans; resulting in an increase in aorta contractions. In the same species, AT stimulates anterior midgut and rectum muscle contractions to mix urine and feces and facilitate the voiding of the rectum. Furthermore, levels of AT in midgut and Malpighian tubules increased in the afternoon when insects are getting ready for nocturnal feeding. In the present study we describe the synergistic effect of AT and serotonin increasing the frequency of contractions of the aorta in Rhodnius prolixus. The basal frequency of contractions of the aorta in the afternoon is higher that the observed during the morning, suggesting the existence of a daily rhythmic activity. The AT receptor is expressed in the rectum, midgut and dorsal vessel, three critical organs involved in post-prandial diuresis. All together these findings provide evidence that AT plays a role as a myoregulatory and cardioacceleratory peptide in R. prolixus.     

Changes in the structure and composition of the extensible cuticle of Rhodnius prolixus through the 5th larval instar

Changes in the composition of the extensible cuticle through the 5th larval instar of Rhodnius prolixus were measured and related to the physiological and developmental state of the larva. Particular attention was paid to the extractable proteins and their characteristics are related to the plasticisation and stretch of the cuticle during the feeding process.There are eight major soluble proteins in the cuticle; three are acidic and five are basic. A model of the cuticle structure held together by ionic and hydrophobic interactions between the constituents is proposed. Polymorphism as a reason for the large number of proteins in ‘soft’ cuticles is refuted on the evidence available.A novel type of transitory cuticle is formed after the feeding of larvae. The role of this cuticle is discussed and a multiple function proposed for it.     

Natriuresis and diuretic hormone synergism in R. prolixus upper Malpighian tubules is inhibited by the anti-diuretic hormone, RhoprCAPA-α2

Insects contain an array of hormones that coordinate the actions of the excretory system to achieve osmotic and ionic balance. In the hematophagous insect, Rhodnius prolixus, two diuretic hormones have been identified, serotonin (5HT) and a corticotropin releasing factor-related peptide (RhoprDH), and both lead to an increase in fluid secretion by Malpighian tubules (MTs). However, only 5HT activates reabsorption by the lower MTs to recover K(+) and Cl(-). An anti-diuretic hormone (RhoprCAPA-α2) is believed to coordinate the cessation of the rapid diuresis following blood meal engorgement. However, the role of RhoprCAPA-α2 on fluid secretion by MTs stimulated by RhoprDH was previously unknown. Here we demonstrate that, unlike the inhibitory effect on 5HT-stimulated secretion by MTs, RhoprCAPA-α2 does not inhibit secretion stimulated by RhoprDH although it does abolish the synergism that occurs between the two diuretic hormones. In addition, we show that the natriuresis elicited by either diuretic hormone is reduced by RhoprCAPA-α2. Using electrophysiological tools, we investigate the possible mechanism by which this complex regulatory pathway is achieved. Analysis of the pH of secreted fluid as well as the triphasic response in transepithelial potential in MTs treated with diuretic hormones, suggests that RhoprCAPA-α2 does not inhibit the V-type H(+) ATPase. Taken together, these results indicate that RhoprCAPA-α2 functions to reduce the rapid diuresis following blood feeding, and in addition, it inhibits the natriuresis associated with diuretic hormone stimulated MTs. This may reflect an important regulatory mechanism related to the slow diuresis that occurs as the K(+)-rich blood cells are digested.     

Biostable insect kinin analogs reduce blood meal and disrupt ecdysis in the blood-gorging Chagas’ disease vector,Rhodnius prolixus

Rhodnius prolixus is a blood-gorging hemipteran that takes blood meals that are approximately 10 times its body weight. This blood meal is crucial for growth and development and is needed to ensure a successful molt into the next instar. Kinins are a multifunctional family of neuropeptides which have been shown to play a role in the control of feeding in a variety of insects. In this study, two biostable Aib-containing kinin analogs were tested to see if they interfere with blood-feeding and subsequent development into the next instar. One of the analogs, 1729 (Ac-R[Aib]FF[Aib]WGa), had no effect on the size of the blood meal or on the subsequent molting of the insect into the next instar. This analog also did not interfere with either short-term or long-term diuresis. The second analog, 1728 ([Aib]FF[Aib]WGa), appeared to be an antifeedant. Insects feeding on blood containing this analog (15 μM) only consumed 60% of the blood meal taken by insects fed on blood without analog. Insects feeding on blood containing 1728 had a slower rate of rapid diuresis (diuresis in the first 3–5 h after feeding) leading to less urine being excreted by 5 days post feeding. The consequence of these effects was that insects fed on 1728 did not molt. This data indicates that the biostable Aib-containing analog 1728 disrupts normal growth and development in the blood-feeding insect, R. prolixus.     

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.     

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.     

Mosquito Aedes aegypti (L.) leucokinin receptor is critical for in vivo fluid excretion post blood feeding

The evolution of the blood feeding adaptation in mosquitoes required hormonal coordination of multiple physiological processes (behavior, digestion, diuresis, oogenesis). The Aedes kinins (leucokinin-like neuropeptides) are involved in post blood feeding physiological processes, having diuretic and myotropic functions. To understand the in vivo contribution of the kinin receptor to overall female post-prandial fluid excretion, RNAi knockdown was followed by fluid secretion assays which proved its fundamental role in rapid diuresis. The Aedes kinin receptor was also localized in several tissues not previously reported in mosquitoes. Results highlight the integrative role of the Aedes kinins in the success of the blood feeding adaptation.     

Serotonin Inhibits Protein Feeding in the Blow Fly, Phormia regina (Meigen)

Serotonin is an important signaling molecule involved in the control of feeding in flies and other animals. In this study, a potential neurohemal release site for serotonin and the effects of exogenous serotonin on protein feeding were examined in the black blow fly, Phormia regina. A dense network of varicose neural processes exhibiting serotonin-like immunoreactivity was identified on the dorsal region of the thoracico-abdominal ganglion in P. regina. This dorsal region of the central nervous system is a likely site for the release of serotonin into the hemolymph. Circulating serotonin may have multiple systemic effects on fly physiology, including modulating or regulating feeding related processes and diuresis. Injections of exogenous serotonin reduced protein meal size in female flies at all doses and at all time points examined within a 24 h period relative to control and saline injected flies. Similar results were observed in serotonin-injected males at 35 min post injection. The injection of 50 μg serotonin resulted in the greatest amount of protein feeding inhibition. Serotonin injected flies also experienced greater weight loss than control or saline-injected flies during the 24 h post-injection period, possibly due to increased diuresis.     

A post-ecdysial plasticization of the abdominal cuticle in Rhodnius

The ecdysis and emergence of fifth instar larvae of Rhodnius prolixus have been closely observed. At the time of ecdysis the cuticle of the head, legs, and wingpads is soft and readily deformed. it does not become sufficiently rigid for normal use until about 90 min later. The cuticle of the abdomen is however hard and inextensible at the time of ecdysis. From about 60 min onward this cuticle undergoes a plasticization; it is maximally extensible at about 180 min, thereafter becoming inextensible again. Unlike the plasticization of the abdominal cuticle which occurs after feeding, this post-ecdysial plasticization is not under direct nervous control. Although it seems that there is some temporal link with the darkening of the cuticle, it is considered unlikely that plasticization is a direct consequence of the tanning process. The significance of this post-ecdysial plasticization is not obvious.     

Changes in the rate of CO2 release following feeding in the insect Rhodnius prolixus

We describe for the first time changes in the rate of CO2 release (as a surrogate of metabolic rate) in the terminal larval stage of the insect Rhodnius prolixus following a blood meal and during the molt leading to the adult stage. These data are presented on a whole-animal basis as well as per gram wet and dry weight. We have also used techniques that allow us to describe the rate of release per gram of actual body tissue (i.e., removing the weight of the remaining bloodmeal in the gut and the metabolically inactive portion of the cuticle). While the metabolic rate of the whole animal rises approximately 10-fold in 15 d following feeding, the rate per gram of dry body mass rises only twofold. We use these data to provide insights into the relative contributions of tissue growth and increases in metabolic intensity to the massive increases in metabolic rate observed in these insects following feeding. Our analyses indicate that the majority of nutrient uptake occurs in the first 4 d following feeding. It is well known in this species that day 4 following feeding is the end of a critical period for the insect in determining whether it will proceed to the next molt. Our results indicate that the insects may be able to make this decision based on nutrients already transported into the body. We examined the "down regulation" of metabolism observed in the latter stages of the molt cycle in this insect. We express these changes on both a per animal and per gram basis and demonstrate that this down regulation extends even into the adult stage before feeding. Using a comparison of the allometric relationships of metabolic rate to mass in insects and ticks, we demonstrate that unfed R. prolixus show a marked decrease in metabolic rate compared to other insects, while fed Rhodnius are similar in metabolic rate to other insects. Rhodnius has a markedly higher metabolic rate (as do all insects) than that found in ticks.     

The properties of the lining membrane of the insect tracheal system

In the surface layer of the lining cuticle of the tracheae of adult Calliphora there is no sign of any waterproofing layer of cuticulin (sclerotin + lipid) as seen in the surface of the general body cuticle. In a few insects: Calliphora adult thorax, Rhodnius adult tracheae serving the ovary, Periplaneta abdominal tracheae, it has been possible to introduce silver hydroxide solution into the lumen of tracheae in the living insect. In each case the silver hydroxide reacted at room temperature with the argentaffin structures in the cuticle, as happens in the soft surface cuticle of Rhodnius larva before moulting or after gentle abrasion. In the thorax of Calliphora the taenidia of the tracheae are stiffened by argentaffin cuticulin. but immediately upon entering the cleft in the flight muscle the taenidia disappear and are replaced by simple folds, so that no stiff taenidia enter the muscle and there is no argentffin material deeper in the flight muscle system.     

Diuretic hormone in adult Rhodnius prolixus: total store and speed of release

Adult Rhodnius take smaller blood meals than do fifth stage larvae and excrete less fluid afterwards. The total amount of diuretic hormone and active precursors that can be extracted from their nervous system is also smaller. However, more diuretic hormone is released in adult than in fifth stage insects when the hormone release sites are depolarized for short periods with potassium-rich solutions. This shows that the adult can release diuretic hormone at a higher rate than can the fifth stage larva, a feature that can be correlated with the larger volume of haemo-lymph which the adult insect has to load with hormone in order to promote diuresis. In vivo , the concentration of diuretic hormone in the haemo-lymph of adult and fifth stage insects rises at the same rate. Detectable amounts of hormone are found in samples of haemolymph from insects which have been feeding for periods as short as 15 s.     

Distribution and activity of a Dippu DH31-like peptide in the large milkweed bug Oncopeltus fasciatus

The milkweed bug, Oncopeltus fasciatus, is a plant feeding hemipteran. While there has been much research done on the neurohormonal control of the post-feeding diuresis in the blood-feeding hemipteran, Rhodnius prolixus, little is known about the control of the post-feeding diuresis in O. fasciatus. One of the neurohormones that may play a role in this rapid diuresis belongs to the calcitonin-like diuretic hormone (DH31) family of insect peptides. In this study we demonstrate the presence of DH31-like immunoreactivity in the central nervous system (CNS) and gut of O. fasciatus 5th instars. As well, DH31-like material was quantified and partially purified from the CNS of 5th instar O. fasciatus using reversed-phase liquid chromatography (RPLC) and monitored with an enzyme-linked immunosorbent assay (ELISA). When tested on O. fasciatus 5th instar Malpighian tubules, DH31-like peptides significantly increased the rate of secretion over saline controls. The results suggest that there is a DH31-like peptide(s) present in the CNS of O. fasciatus and that this peptide may play a role in the control of Malpighian tubule secretion.     

The crystal structure of nitrophorin 2. A trifunctional antihemostatic protein from the saliva of Rhodnius prolixus

Nitrophorin 2 (NP2) (also known as prolixin-S) is a salivary protein that transports nitric oxide, binds histamine, and acts as an anticoagulant during blood feeding by the insect Rhodnius prolixus. The 2.0-A crystal structure of NP2 reveals an eight-stranded antiparallel beta-barrel containing a ferric heme coordinated through His(57), similar to the structures of NP1 and NP4. All four Rhodnius nitrophorins transport NO and sequester histamine through heme binding, but only NP2 acts as an anticoagulant. Here, we demonstrate that recombinant NP2, but not recombinant NP1 or NP4, is a potent anticoagulant; recombinant NP3 also displays minor activity. Comparison of the nitrophorin structures suggests that a surface region near the C terminus and the loops between beta strands B-C and E-F is responsible for the anticoagulant activity. NP2 also displays larger NO association rates and smaller dissociation rates than NP1 and NP4, which may result from a more open and more hydrophobic distal pocket, allowing more rapid solvent reorganization on ligand binding. The NP2 protein core differs from NP1 and NP4 in that buried Glu(53), which allows for larger NO release rates when deprotonated, hydrogen bonds to invariant Tyr(81). Surprisingly, this tyrosine lies on the protein surface in NP1 and NP4.     

Perimicrovillar membranes promote hemozoin formation into Rhodnius prolixus midgut

Rhodnius prolixus is a hematophagous insect that ingests large quantities of blood in each blood-feeding session. This ingested blood provides important nutrients to sustain the insect's oogenesis and metabolic pathways. During the digestive process, however, huge amounts of heme are generated as a consequence of the hemoglobin breakdown. Heme is an extremely dangerous molecule, since it can generate reactive oxygen species in the presence of oxygen that impair the normal metabolism of the insect. Part of the hemoglobin-derived heme can associate with the perimicrovillar membranes (PMM) in the gut lumen of R. prolixus; in this study we demonstrate the participation of the PMM in a heme detoxification process. These membranes were able to successfully induce heme aggregation into hemozoin (Hz). Heme aggregation was not dependent on the erythrocyte membranes, since the contribution of these membranes to the process was negligible, demonstrating that the ability to induce heme aggregation is a feature of the PMM, possibly representing a pre-adaptation of the hemipterans to feeding on blood.