Mechanisms controlling haemolymph circulation under resting conditions in the Chagas disease vector Rhodnius prolixus.

Chagas disease vectors can ingest several times their own volume in blood with each meal. This ad libitum feeding causes an intense process of diuresis, inducing the insect to eliminate a large quantity of urine and faeces. To ensure diuresis, the speed of circulation of the haemolymph is increased. The Triatominae circulatory system is quite simple, including the dorsal vessel, which pumps haemolymph in an anterograde direction. The return is caused by peristaltic contractions of the anterior midgut. Triatominae insects can spend several weeks without feeding, meaning that most of the time, the insect is in a resting condition. Although the mechanisms controlling the circulation of the haemolymph during post-prandial diuresis have been largely analysed, the mechanisms controlling it during resting conditions are poorly understood. In this study, we analysed several canonical pathways (i.e. L-type VGCC, GPCR, RyR, IP3R) and a novel system represented by the recently characterized Piezo proteins. Our results show that during the resting condition, haemolymph circulation depends on a cross-talk between myogenic activity, inhibitory and stimulatory cellular messengers, and Piezo proteins. This report also unveils for the first time the existence of a putative Piezo protein in Hemiptera.

RhoprCAPA-2 acts as a gonadotropin regulating reproduction in adult female, Rhodnius prolixus.

CAPA peptides play diverse roles in insects, modulating muscle contraction, regulating fluid balance, and reproduction. In Rhodnius prolixus, a hematophagous insect and a vector for human Chagas disease, three CAPA peptides are encoded by the capability gene, including RhoprCAPA-1, RhoprCAPA-2, and RhoprCAPA-PK-1. RhoprCAPA-2 is an anti-diuretic hormone in R. prolixus. Here, we explore the involvement of RhoprCAPA-2 in reproduction in adult female R. prolixus. Double-label immunohistochemistry reveals co-localization of RhoprCAPA-2-like and the glycoprotein hormone (GPA2/GPB5) subunit GPB5-like immunoreactivity in neurosecretory cells in the mesothoracic ganglionic mass and in their neurohemal sites, suggesting these peptides can be co-released to regulate physiological processes. qPCR analysis reveals changes in transcript expression levels of the RhoprCAPA receptor (CAPAR) in the fat body and reproductive tissues after feeding in adult female R. prolixus. RNA interference-mediated knockdown of CAPAR transcript decreases egg production and reduces hatching rate and survival rate in female R. prolixus. Downregulation of CAPAR decreases vitellogenin RhoprVg1 transcript expression in the fat body and deceases its receptor RhoprVgR transcript level in the ovaries; accompanied by a reduction in vitellogenin content in the fat body and hemolymph. Incubation of fat body and ovaries in vitro with RhoprCAPA-2 increases RhoprVg1 transcript expression in the fat body, vitellogenin content in the fat body culture medium, and increases RhoprVgR transcript in the ovaries. These findings implicate the CAPA signaling pathway in reproduction, with RhoprCAPA-2 acting as a gonadotropin in adult female R. prolixus.

Deficiency of ULK1/ATG1 in the follicle cells disturbs ER homeostasis and causes defective chorion deposition in the vector Rhodnius prolixus.

In insects, synthesis and deposition of the chorion (eggshell) are performed by the professional secretory follicle cells (FCs) that surround the oocytes in the course of oogenesis. Here, we found that ULK1/ATG1, an autophagy-related protein, is highly expressed in the FCs of the Chagas-Disease vector Rhodnius prolixus, and that parental RNAi silencing of ULK1/ATG1 results in oocytes with abnormal chorion ultrastructure and FCs presenting expanded rough ER membranes as well as increased expression of the ER chaperone BiP3, both indicatives of ER stress. Silencing of LC3/ATG8, another essential autophagy protein, did not replicate the ULK1/ATG1 phenotypes, whereas silencing of SEC16A, a known partner of the noncanonical ULK1/ATG1 function in the ER exit sites phenocopied the silencing of ULK1/ATG1. Our findings point to a cooperated function of ULK1/ATG1 and SEC16A in the FCs to complete choriogenesis and provide additional in vivo phenotype-based evidence to the literature of the role of ULK1/ATG1 in the ER in a professional secretory cell.

Deciphering the role of Rhodnius prolixus CYP4G genes in straight and methyl-branched hydrocarbon formation and in desiccation tolerance.

Insect cuticle hydrocarbons are involved primarily in waterproofing the cuticle, but also participate in chemical communication and regulate the penetration of insecticides and microorganisms. The last step in insect hydrocarbon biosynthesis is carried out by an insect-specific cytochrome P450 of the 4G subfamily (CYP4G). Two genes (CYP4G106 and CYP4G107) have been reported in the triatomines Rhodnius prolixus and Triatoma infestans. In this work, their molecular and functional characterization is carried out in R. prolixus, and their relevance to insect survival is assessed. Both genes are expressed almost exclusively in the integument and have an expression pattern dependent on the developmental stage and feeding status. CYP4G106 silencing diminished significantly the straight-chain hydrocarbon production while a significant reduction - mostly of methyl-branched chain hydrocarbons - was observed after CYP4G107 silencing. Molecular docking analyses using different aldehydes as hydrocarbon precursors predicted a better fit of straight-chain aldehydes with CYP4G106 and methyl-branched aldehydes with CYP4G107. Survival bioassays exposing the silenced insects to desiccation stress showed that CYP4G107 is determinant for the waterproofing properties of the R. prolixus cuticle. This is the first report on the in vivo specificity of two CYP4Gs to make mostly straight or methyl-branched hydrocarbons, and also on their differential contribution to insect desiccation.

Triatomines of the Genus Rhodnius Do Not Mark Shelters with Feces.

Aggregation to volatile compounds emitted by feces has been demonstrated for several triatomine species. This signal guides the insects to suitable places that offer physical protection while providing contact with conspecifics. Though the use of aggregation marks has also been reported for Rhodnius spp., it is still unclear whether feces really cause these insects to aggregate inside shelters. In two-choice assays using artificial shelters, we found that refuges associated with a blend of synthetic compounds based on volatiles released by the feces of some triatomine species and reported to be attractive to several species, did not induce shelter choice in Rhodnius prolixus Stål, 1859. In addition, we show that refuges associated with feces of conspecifics did not induce shelter choice in R. prolixus, R. robustus Larrousse, 1927, R. neglectus Lent, 1954, and R. ecuadoriensis Lent and León, 1958. In contrast, as expected, control experiments with Triatoma infestans (Klug, 1834), another triatomine known to aggregate in shelters marked with feces, definitely showed preference for shelters containing feces of conspecifics. Our results clearly show that volatile signals associated with feces do not mediate shelter choice in Rhodnius spp. As a consequence, a paradigm shift will be necessary and, consequently, ab ovo investigations on the clues inducing aggregation behavior in these species.

The neuropeptide CCHamide2 regulates diuresis in the Chagas disease vector Rhodnius prolixus.

Given that hematophagous insects ingest large quantities of blood in a single meal, they must undergo a rapid post-prandial diuresis in order to maintain homeostasis. In the kissing bug Rhodnius prolixus (Hemiptera: Reduviidae), the coordinated activity of the Malpighian tubules and anterior midgut maintains water and ion balance during the post-prandial diuresis. Three to four hours after the meal, the diuretic process finishes, and the animal enters an antidiuretic state to ensure water conservation until the next blood intake. The diuretic and antidiuretic processes are tightly regulated by serotonin and neuropeptides in this insect. In the present work, we report that the neuropeptide precursor CCHamide2 is involved in the regulation of the post-prandial diuresis in R. prolixus Our results suggest a dual effect of RhoprCCHamide2 peptide, enhancing the serotonin-induced secretion by Malpighian tubules, and inhibiting serotonin-induced absorption across the anterior midgut. To our knowledge, this is the first report of a hormone presenting opposite effects in the two osmoregulatory organs (i.e. midgut and Malpighian tubules) in insects, probably reflecting the importance of a well-tuned diuretic process in hematophagous insects during different moments after the blood meal.

The involvement of Rhopr-CRF/DH in feeding and reproduction in the blood-gorging insect Rhodnius prolixus.

Rhodnius prolixus is a blood-gorging insect and a vector for human Chagas disease. The insect transmits the disease following feeding, when it excretes urine and feces contaminated with the Trypanosoma cruzi parasite. A corticotropin-releasing factor-like peptide acts as a diuretic hormone in R. prolixus (Rhopr-CRF/DH); however, its distribution throughout the insect's central nervous system (CNS) and the expression of its receptor in feeding-related tissue as well as the female reproductive system suggests a multifaceted role for the hormone beyond that of diuresis. Here we investigate the involvement of Rhopr-CRF/DH in feeding and reproduction in R. prolixus. Immunohistochemistry of the CNS showed diminished CRF-like staining in neurosecretory cells (NSCs) of the mesothoracic ganglionic mass (MTGM) immediately following feeding, and partial restocking of those same cells two hours later, indicating Rhopr-CRF/DH stores in this regions are involved in feeding. The results of the temporal qPCR analysis were consistent with the immunohistochemical findings, showing an increase in Rhopr-CRF/DH transcript expression in the MTGM immediately after feeding, presumably capturing the restocking of Rhopr-CRF/DH in the lateral NSCs following release of the peptide during feeding. Elevating haemolymph Rhopr-CRF/DH titres by injection of Rhopr-CRF/DH prior to feeding resulted in the intake of a significantly smaller blood meal in 5th instars and adults without an apparent effect on the rate of short-term diuresis. When adult females were injected with Rhopr-CRF/DH, they also produced and laid significantly fewer eggs. Finally, in vitro oviduct contraction assays illustrate that Rhopr-CRF/DH inhibits the amplitude of contractions of the lateral oviducts, highlighting a potential mechanism via which the hormone diminishes reproductive capacity. To conclude, the study of the Rhopr-CRF/DH pathway, its components and mechanisms of action, has implications for vector control by highlighting targets to alter feeding, diuresis, and reproduction of this disease vector.

Rhodnius prolixus: From classical physiology to modern developmental biology.

The hemiptera Rhodnius prolixus is a blood-feeding insect and a primary vector of Trypanosoma cruzi, the etiological agent of the Chagas disease. Over the past century, Rhodnius has been the subject of intense investigations, which have contributed to unveil important aspects of metabolism and physiology in insects. Recent technological innovations are helping dissect the genetic and molecular underpinnings of Rhodnius embryogenesis and organogenesis, thus fostering the use of this important species in the fields of developmental and evolutionary biology. Rhodnius represents also an excellent system to study development under stressful conditions, since the embryo must develop in the presence of a large amount of blood-derived reactive oxygen species. With a recently sequenced genome, small among other Hemiptera, and the identification of basic elements for all classical development pathways, functional studies in this species are revealing novel aspects of insect development and evolution. Here we review early studies on this model insect and how this paved the way for recent functional studies using the kissing bug.

Using eco-physiological traits to understand the realized niche: the role of desiccation tolerance in Chagas disease vectors.

Small ectotherms, such as insects, with high surface area-to-volume ratios are usually at risk of dehydration in arid environments. We hypothesize that desiccation tolerance in insects could be reflected in their distribution, which is limited by areas with high relative values of water vapor pressure deficit (VPD) (e.g., hot and dry). The main goal of this study was to explore whether incorporation of eco-physiological traits such as desiccation tolerance in arid environments can improve our understanding of species distribution models (SDM). We use a novel eco-physiological approach to understand the distribution and the potential overlap with their fundamental niche in triatomine bugs, Chagas disease vectors. The desiccation dimension for T. infestans, T. delpontei, T. dimidiata, and T. sordida niches seems to extend to very dry areas. For T. vitticeps, xeric areas seem to limit the geographical range of their realized niche. The maximum VPD limits the western and southern distributions of T. vitticeps, T. delpontei, and T. patagonica. All species showed high tolerance to desiccation with survival times (35 °C-RH ~ 15%) ranging from 24 to 38 days, except for T. dimidiata (9 days), which can be explained by a higher water-loss rate, due to a higher cuticular permeability along with a higher critical water content. This approach indicates that most of these triatomine bugs could be exploiting the dryness dimension of their fundamental niche. Incorporating such species-specific traits in studies of distribution, range, and limits under scenarios of changing climate could enhance predictions of movement of disease-causing vectors into novel regions.

Differences in competitive ability for the occupancy of shelters in triatomines.

Triatomines (Hemiptera: Reduviidae: Triatominae) are nocturnal blood-sucking insects. During daylight hours they remain in an akinetic state inside their shelters, whereas at dusk they become active and move outside. When they are outside their shelters during the photophase, triatomines are vulnerable to diurnal predators and the period just before dawn is critical to their survival. This work analyses the existence of competitive interactions involved in the occupancy of shelters by triatomines. Behavioural assays were performed in which nymphs of different stages, nutritional status or species were released in an experimental arena containing a space-limited artificial shelter. The proportions of individuals occupying the shelter during the photophase were quantified to estimate the competitive abilities of each stage and species. Intraspecific comparisons showed higher levels of shelter occupancy for fourth over fifth instars and fed over unfed nymphs of Triatoma infestans. Interspecific comparisons showed higher rates of shelter occupancy for Triatoma sordida in comparison with T. infestans, and for T. infestans over Rhodnius prolixus. Arrival order was also relevant to determining shelter occupancy levels: early arrival was advantageous in comparison with later arrival. The study of intra- and interspecific competitive interactions for shelter occupancy provides relevant information about colonization and recolonization processes in the natural environments of triatomines.

Haematophagy is costly: respiratory patterns and metabolism during feeding in Rhodnius prolixus.

Feeding on the blood of vertebrates is a risky task for haematophagous insects and it can be reasonably assumed that it should also be costly in terms of energetic expenditure. Blood circulates inside vessels and it must be pumped through narrow tubular stylets to be ingested. We analysed the respiratory pattern and the energetic cost of taking a blood meal in Rhodnius prolixus using flow-through and stop-flow respirometry to measure carbon dioxide emission, oxygen consumption and water loss before and during feeding. We observed an increase of up to 17-fold in the metabolic rate during feeding and a change in the respiratory pattern, which switched from a discontinuous cyclic pattern during resting to a continuous pattern when the insects started to feed, remaining in this condition unchanged for several hours. The energetic cost of taking a meal was significantly higher when bugs fed on a living host, compared with feeding on an artificial feeder. No differences were observed between feeding on blood or on saline solution in vitro, revealing that the substrate for feeding (vessels versus membrane) and not the nature of the fluid was responsible for such a difference in the energetic cost. Water loss significantly increased during feeding, but did not vary with feeding method or type of food. The mean respiratory quotient in resting bugs was 0.83, decreasing during feeding to 0.52. These data constitute the first metabolic measures of an insect during blood feeding and provide the first insights into the energetic expenditure associated with haematophagy.

Temperature and parasite life-history are important modulators of the outcome of Trypanosoma rangeli-Rhodnius prolixus interactions.

Trypanosoma rangeli is a protozoan parasite, which does not cause disease in humans, although it can produce different levels of pathogenicity to triatomines, their invertebrate hosts. We tested whether infection imposed a temperature-dependent cost on triatomine fitness using T. rangeli with different life histories. Parasites cultured only in liver infusion tryptose medium (cultured) and parasites exposed to cyclical passages through mice and triatomines (passaged) were used. We held infected insects at four temperatures between 21 and 30 °C and measured T. rangeli growth in vitro at the same temperatures in parallel. Overall, T. rangeli infection induced negative effects on insect fitness. In the case of cultured infection, parasite effects were temperature-dependent. Intermoult period, mortality rates and ecdysis success were affected in those insects exposed to lower temperatures (21 and 24 °C). For passaged-infected insects, the effects were independent of temperature, intermoult period being prolonged in all infected groups. Trypanosoma rangeli seem to be less tolerant to higher temperatures since cultured-infected insects showed a reduction in the infection rates and passaged-infected insects decreased the salivary gland infection rates in those insects submitted to 30 °C. In vitro growth of T. rangeli was consistent with these results.

Trypanosoma cruzi-Trypanosoma rangeli co-infection ameliorates negative effects of single trypanosome infections in experimentally infected Rhodnius prolixus.

Trypanosoma cruzi, causative agent of Chagas disease, co-infects its triatomine vector with its sister species Trypanosoma rangeli, which shares 60% of its antigens with T. cruzi. Additionally, T. rangeli has been observed to be pathogenic in some of its vector species. Although T. cruzi-T. rangeli co-infections are common, their effect on the vector has rarely been investigated. Therefore, we measured the fitness (survival and reproduction) of triatomine species Rhodnius prolixus infected with just T. cruzi, just T. rangeli, or both T. cruzi and T. rangeli. We found that survival (as estimated by survival probability and hazard ratios) was significantly different between treatments, with the T. cruzi treatment group having lower survival than the co-infected treatment. Reproduction and total fitness estimates in the T. cruzi and T. rangeli treatments were significantly lower than in the co-infected and control groups. The T. cruzi and T. rangeli treatment group fitness estimates were not significantly different from each other. Additionally, co-infected insects appeared to tolerate higher doses of parasites than insects with single-species infections. Our results suggest that T. cruzi-T. rangeli co-infection could ameliorate negative effects of single infections of either parasite on R. prolixus and potentially help it to tolerate higher parasite doses.

A role for host-parasite interactions in the horizontal transfer of transposons across phyla.

Horizontal transfer (HT), or the passage of genetic material between non-mating species, is increasingly recognized as an important force in the evolution of eukaryotic genomes. Transposons, with their inherent ability to mobilize and amplify within genomes, may be especially prone to HT. However, the means by which transposons can spread across widely diverged species remain elusive. Here we present evidence that host-parasite interactions have promoted the HT of four transposon families between invertebrates and vertebrates. We found that Rhodnius prolixus, a triatomine bug feeding on the blood of various tetrapods and vector of Chagas' disease in humans, carries in its genome four distinct transposon families that also invaded the genomes of a diverse, but overlapping, set of tetrapods. The bug transposons are approximately 98% identical and cluster phylogenetically with those of the opossum and squirrel monkey, two of its preferred mammalian hosts in South America. We also identified one of these transposon families in the pond snail Lymnaea stagnalis, a cosmopolitan vector of trematodes infecting diverse vertebrates, whose ancestral sequence is nearly identical and clusters with those found in Old World mammals. Together these data provide evidence for a previously hypothesized role of host-parasite interactions in facilitating HT among animals. Furthermore, the large amount of DNA generated by the amplification of the horizontally transferred transposons supports the idea that the exchange of genetic material between hosts and parasites influences their genomic evolution.

Allatostatin-C antagonizes the synergistic myostimulatory effect of allatotropin and serotonin in Rhodnius prolixus (Stal).

Haematophagous insects can ingest large quantities of blood in a single meal producing a large quantity of urine in the following hours to eliminate the excess of water and mineral ions incorporated. The excretory activity of the Malpighian tubules is facilitated by an increase in haemolymph circulation as a result of the intensification of aorta contractions, combined with an increase of anterior midgut peristaltic waves. We have recently shown that haemolymph circulation during post-prandial diuresis is modulated by the synergistic activity of allatotropin (AT) and serotonin, resulting in an increase in aorta and crop contraction rates. In the present study we describe the antagonistic effect of allatostatin-C (AST-C) on the increase of aorta frequency of contractions induced by serotonin/AT in Rhodnius prolixus. The administration of AST-C counteracted the increase in the frequency induced by the treatment with serotonin/AT, but did not affect the increase in frequency induced by the administration of serotonin alone, suggesting that AST-C is altering the synergism between serotonin and AT. Furthermore, the administration of AST-C during post-prandial diuresis decreases the number of peristaltic waves of the anterior midgut. The AST-C putative receptor is expressed in the hindgut, midgut and dorsal vessel, three critical organs involved in post-prandial diuresis. All together these findings provide evidence that AST-C plays a key role as a myoregulatory and cardioregulatory peptide in R. prolixus.

Circadian control of prothoracicotropic hormone release in an adult insect and the induction of its rhythmicity by light cues.

The insect neuropeptide prothoracicotropic hormone (PTTH) is a critical regulator of larval development. We recently demonstrated that PTTH is also present in adult Rhodnius prolixus and is released by adult brains in vitro with a clear daily rhythm during egg development. Here, we employ a well-established in vitro bioassay, to show that the daily rhythm of PTTH release by brains in vitro is under circadian control since it persists in aperiodic conditions with a free running period of around 24h that is temperature compensated. Prolonged exposure (3weeks) of insects to continuous constant light (LL) completely eliminated PTTH release. Subsequent transfer of such insects from LL to constant darkness (DD) rapidly induced rhythmic PTTH release, indicating that the circadian rhythm of PTTH release is induced by photic cues. Western analysis identified PTTH in the adult hemolymph, suggesting that PTTH acts as a functional neurohormone in the adult insect. Dot blot analysis revealed that PTTH levels in the hemolymph also cycled with a daily rhythm that persisted in DD and was synchronous with the rhythm of PTTH release by brains in vitro. We conclude that the previously documented photosensitive clock in the brain regulates rhythmic PTTH release and thus generates the rhythm seen in the hemolymph. These results emphasize the importance of rhythmic PTTH release in the adult insect and support a role for PTTH in adult physiology and possibly within the adult circadian system.

A phospholipase A2 gene is linked to Jack bean urease toxicity in the Chagas' disease vector Rhodnius prolixus.

BACKGROUND: Ureases are multifunctional enzymes that display biological activities independent of their enzymatic function, including exocytosis induction and insecticidal effects. The hemipteran Rhodnius prolixus is one of the known susceptible models for this toxicity. It has been shown that Jack bean urease (JBU) has deleterious effects on R. prolixus, and these effects are modulated by eicosanoids, which are synthesized in a cascade involving phospholipase A2 (PLA2) enzymes. METHODS: R. prolixus genome was screened for putative PLA2s and matching transcripts were cloned. Predicted amino acid sequences were analyzed and transcript distribution among tissues was determined by qPCR. RNAi techniques were used and subsequent JBU toxicity assays were performed. RESULTS: Two PLA2 genes were identified, Rhopr-PLA2III and Rhopr-PLA2XII. The transcripts are widely distributed in the tissues but at different levels. The analyses fit the putative proteins into groups III and XII of secretory PLA2s. After 70% of Rhopr-PLA2XII expression was knocked down, JBU's toxicity was decreased by more than 50% on 5th instars R. prolixus. CONCLUSIONS: Rhopr-PLA2XII gene is linked to JBU's toxic effect in R. prolixus and our findings support previous studies demonstrating that eicosanoids modulate this toxicity. GENERAL SIGNIFICANCE: Besides identifying and characterizing two PLA2 genes in the major Chagas' disease vector R. prolixus, we have shown that the potent toxicity of JBU is linked to one of these genes. Our results contribute to the general comprehension of urease's mechanisms of action in insects, and, potentially, to studies on the control of the Chagas' disease parasite transmission.

The effect of convection on infrared detection by antennal warm cells in the bloodsucking bug Rhodnius prolixus.

Previous work revealed that bloodsucking bugs can discriminate between oscillating changes in infrared (IR) radiation and air temperature (T) using two types of warm cells located in peg-in-pit sensilla and tapered hairs (Zopf LM, Lazzari CR, Tichy H. J Neurophysiol 111: 1341-1349, 2014). These two stimuli are encoded and discriminated by the response quotient of the two warm cell types. IR radiation stimulates the warm cell in the peg-in-pit sensillum more strongly than that in the tapered hair. T stimuli evoke the reverse responses; they stimulate the latter more strongly than the former. In nature, IR and T cues are always present with certain radiation intensities and air temperatures, here referred to as background IR radiation and background T. In this article, we found that the response quotient permits the discrimination of IR and T oscillations even in the presence of different backgrounds. We show that the two warm cells respond well to IR oscillations if the background T operates by natural convection but poorly at forced convection, even if the background T is higher than at natural convection. Background IR radiation strongly affects the responses to T oscillations: the discharge rates of both warm cells are higher the higher the power of the IR background. We compared the warm cell responses with the T measured inside small model objects shaped like a cylinder, a cone, or a disc. The experiments indicate that passive thermal effects of the sense organs rather than intrinsic properties of the sensory cells are responsible for the observed results.

Identification, characterization and expression of a receptor for the unusual myosuppressin in the blood-feeding bug, Rhodnius prolixus.

Myosuppressins are a family of the FMRFamide-like peptides. They have been characterized in many insects and shown to inhibit visceral muscle contraction. Rhodnius prolixus possesses an unusual myosuppressin in that the typical FLRFamide C-terminal motif is unique and ends with FMRFamide. In the present study, we isolated the cDNA sequence for the R. prolixus receptor for this unusual myosuppressin (RhoprMSR). Quantitative PCR indicates high relative transcript expression of RhoprMSR in the central nervous system and also supports the previously described physiological effects of RhoprMS on the digestive system, with expression of the RhoprMSR transcript in the midgut and hindgut. Expression of the RhoprMSR transcript was also found in the female and male reproductive system of 5th instar nymphs, with transcript expression greater in the female reproductive tissues. No expression was found in the salivary glands or Malpighian tubules. A functional receptor expression assay confirmed that the cloned RhoprMSR is indeed activated by RhoprMS (half maximum effective concentration = 42.7 nM). Structure-activity studies based upon both functional receptor assays and physiological assays showed the importance of the HVFMRFamide moiety, as further N-terminal truncation removed all activity.

Circadian modulation of learning ability in a disease vector insect, Rhodnius prolixus.

Despite the drastic consequences it may have on the transmission of parasites, the ability of disease vectors to learn and retain information has just begun to be characterised. The kissing bug Rhodnius prolixus, a vector of Chagas disease, is an excellent model, particularly because conditioning the proboscis extension response (PER) constitutes a valuable paradigm to study their cognitive abilities under carefully controlled conditions. Another characteristic of these bugs is the temporal organisation of their different activities in a bimodal endogenous daily rhythm. This offers the opportunity to address the implication of the circadian system in learning and memory. Using aversive conditioning of the PER, we tested whether the ability of kissing bugs to learn and remember information varies during the day. We found that bugs perform well during the night, but not during the day: their ability to acquire information - but not their ability to retrieve it - is modulated by time. When the bugs were kept under constant conditions in order to analyse the origin of this rhythm, the rhythm continued to free run, showing its endogenous and truly circadian nature. These results are the first to evince the effect of the circadian system on the learning abilities of disease vectors and one of the few in insects in general.