Unveiling the Peptidase Network Orchestrating Hemoglobin Catabolism in Rhodnius prolixus.

Chagas disease is transmitted to humans by obligatory hematophagous insects of Triatominae subfamily, which feeds on various hosts to acquire their nutritional sustenance derived from blood proteins. Hemoglobin (Hb) digestion is a pivotal metabolic feature of triatomines, representing a key juncture in their competence toward Trypanosoma cruzi; however, it remains poorly understood. To explore the Hb digestion pathway in Rhodnius prolixus, a major Chagas disease vector, we employed an array of approaches for activity profiling of various midgut-associated peptidases using specific substrates and inhibitors. Dissecting the individual contribution of each peptidase family in Hb digestion has unveiled a predominant role played by aspartic proteases and cathepsin B-like peptidases. Determination of peptidase-specific cleavage sites of these key hemoglobinases, in conjunction with mass spectrometry-based identification of in vivo Hb-derived fragments, has revealed the intricate network of peptidases involved in the Hb digestion pathway. This network is initiated by aspartic proteases and subsequently sustained by cysteine proteases belonging to the C1 family. The process is continued simultaneously by amino and carboxypeptidases. The comprehensive profiling of midgut-associated aspartic proteases by quantitative proteomics has enabled the accurate revision of gene annotations within the A1 family of the R. prolixus genome. Significantly, this study also serves to illuminate a potentially important role of the anterior midgut in blood digestion. The expanded repertoire of midgut-associated proteases presented in this study holds promise for the identification of novel targets aimed at controlling the transmission of Chagas disease.

Export of heme by the feline leukemia virus C receptor regulates mitochondrial biogenesis and redox balance in the hematophagous insect Rhodnius prolixus.

Heme is a prosthetic group of proteins involved in vital physiological processes. It participates, for example, in redox reactions crucial for cell metabolism due to the variable oxidation state of its central iron atom. However, excessive heme can be cytotoxic due to its prooxidant properties. Therefore, the control of intracellular heme levels ensures the survival of organisms, especially those that deal with high concentrations of heme during their lives, such as hematophagous insects. The export of heme initially attributed to the feline leukemia virus C receptor (FLVCR) has recently been called into question, following the discovery of choline uptake by the same receptor in mammals. Here, we found that RpFLVCR is a heme exporter in the midgut of the hematophagous insect Rhodnius prolixus, a vector for Chagas disease. Silencing RpFLVCR decreased hemolymphatic heme levels and increased the levels of intracellular dicysteinyl-biliverdin, indicating heme retention inside midgut cells. FLVCR silencing led to increased expression of heme oxygenase (HO), ferritin, and mitoferrin mRNAs while downregulating the iron importers Malvolio 1 and 2. In contrast, HO gene silencing increased FLVCR and Malvolio expression and downregulated ferritin, revealing crosstalk between heme degradation/export and iron transport/storage pathways. Furthermore, RpFLVCR silencing strongly increased oxidant production and lipid peroxidation, reduced cytochrome c oxidase activity, and activated mitochondrial biogenesis, effects not observed in RpHO-silenced insects. These data support FLVCR function as a heme exporter, playing a pivotal role in heme/iron metabolism and maintenance of redox balance, especially in an organism adapted to face extremely high concentrations of heme.

Local age-dependent neuromodulation in Rhodnius prolixus antennae.

Kissing bugs do not respond to host cues when recently molted and only exhibit robust host-seeking several days after ecdysis. Behavioral plasticity has peripheral correlates in antennal gene expression changes through the week after ecdysis. The mechanisms regulating these peripheral changes are still unknown, but neuropeptide, G-protein coupled receptor, nuclear receptor, and takeout genes likely modulate peripheral sensory physiology. We evaluated their expression in antennal transcriptomes along the first week postecdysis of Rhodnius prolixus 5th instar larvae. Besides, we performed clustering and co-expression analyses to reveal relationships between neuromodulatory (NM) and sensory genes. Significant changes in transcript abundance were detected for 50 NM genes. We identified 73 sensory-related and NM genes that were assigned to nine clusters. According to their expression patterns, clusters were classified into four groups: two including genes up or downregulated immediately after ecdysis; and two with genes with expression altered at day 2. Several NM genes together with sensory genes belong to the first group, suggesting functional interactions. Co-expression network analysis revealed a set of genes that seem to connect with sensory system maturation. Significant expression changes in NM components were described in the antennae of R. prolixus after ecdysis, suggesting that a local NM system acts on antennal physiology. These changes may modify the sensitivity of kissing bugs to host cues during this maturation interval.

Halloween genes are expressed with a circadian rhythm during development in prothoracic glands of the insect RHODNIUS PROLIXUS.

We analyse the developmental and circadian profiles of expression of the genes responsible for ecdysteroidogenesis (Halloween genes) in the PGs of Rhodnius prolixus throughout larval-adult development. Extensive use of in vitro techniques enabled multiple different parameters to be measured in individual PGs. Expression of disembodied and spook closely paralleled the ecdysteroid synthesis of the same PGs, and the ecdysteroid titre in vivo, but with functionally significant exceptions. Various tissues other than PGs expressed one, both or neither genes. Both gonads express both genes in pharate adults (larvae close to ecdysis). Both genes were expressed at low, but significant, levels in UF Rhodnius, raising questions concerning how developmental arrest is maintained in UF animals. IHC confirmed the subcellular localisation of the coded proteins. Gene knockdown suppressed transcription of both genes and ecdysteroid synthesis, with spook apparently regulating the downstream gene disembodied. Transcription of both genes occurred with a daily rhythm (with peaks at night) that was confirmed to be under circadian control using aperiodic conditions. The complex behaviour of the rhythm in LL implied two anatomically distinct oscillators regulate this transcription rhythm. First, the circadian clock in the PGs and second, the circadian rhythm of of Rhodnius PTTH which is released rhythmically from the brain under control of the circadian clock therein, both of which were described previously. We conclude ecdysteroidogenesis in Rhodnius PGs employs a similar pathway as other insects, but its control is complex, involving mechanisms both within and outside the PGs.

Functional characterization of the kinin receptor in the Chagas disease vector Rhodnius prolixus; activity of native kinins and potent biostable Aib-containing insect kinin analogs.

The causative agent for Chagas disease, Trypanosoma cruzi, is transmitted to a human host in the urine/feces of the kissing bug, Rhodnius prolixus, following blood feeding. Kinins are important chemical messengers in the overall control of blood feeding physiology in R. prolixus, including hindgut contractions and excretion. Thus, disruption in kinin signaling would have damaging consequences to the insect but also interfere with the transmission of Chagas Disease. Here, a heterologous functional receptor assay was used to confirm the validity of the previously cloned putative kinin G-protein-coupled receptor, RhoprKR, in Rhodnius prolixus. Three native R. prolixus kinins were chosen for analysis; two possessing the typical kinin WGamide C-terminal motif and one that possesses an atypical C-terminal WAamide. All three are potent (EC50 values in the nM range), with high efficacy, on CHO-K1-aeq cells expressing the RhoprKR, thereby confirming ligand binding. Members of three other R. prolixus peptide families, which are also myotropins (tachykinins, pyrokinins and sulfakinins) elicited little or no response. In addition, this heterologous receptor assay was used to test characteristics of kinin mimetics previously tested on tick and mosquito kinin receptors. Five α-aminoisobutyric acid (Aib) containing analogs were tested, and four found to have considerably higher potencies than the native kinins, with EC50 values in the pM range. Interestingly, adding Aib to the atypical WAamide kinin improves its EC50 value from 2 nM to 39 pM. Biostable kinin analogs may prove useful leads for novel pest control strategies. Since T. cruzi is transmitted to a human host in the urine/feces after blood feeding, disruption in kinin signaling would also interfere with the transmission of Chagas Disease.

Octopamine is required for successful reproduction in the classical insect model, Rhodnius prolixus.

In insects, biogenic amines function as neurotransmitters, neuromodulators, and neurohormones, influencing various behaviors, including those related to reproduction such as response to sex pheromones, oogenesis, oviposition, courtship, and mating. Octopamine (OA), an analog of the vertebrate norepinephrine, is synthesized from the biogenic amine tyramine by the enzyme tyramine ß-hydroxylase (TßH). Here, we investigate the mechanisms and target genes underlying the role of OA in successful reproduction in females of Rhodnius prolixus, a vector of Chagas disease, by downregulating TßH mRNA expression (thereby reducing OA content) using RNA interference (RNAi), and in vivo and ex vivo application of OA. Injection of females with dsTßH impairs successful reproduction at least in part, by decreasing the transcript expression of enzymes involved in juvenile hormone biosynthesis, the primary hormone for oogenesis in R. prolixus, thereby interfering with oogenesis, ovulation and oviposition. This study offers valuable insights into the involvement of OA for successful reproduction in R. prolixus females. Understanding the reproductive biology of R. prolixus is crucial in a medical context for controlling the spread of the disease.

Perimicrovillar membrane assembly: the fate of phospholipids synthesised by the midgut of Rhodnius prolixus

In this study, we describe the fate of fatty acids that are incorporated from the lumen by the posterior midgut epithelium of Rhodnius prolixus and the biosynthesis of lipids. We also demonstrate that neutral lipids (NL) are transferred to the haemolymphatic lipophorin (Lp) and that phospholipids remain in the tissue in which they are organised into perimicrovillar membranes (PMMs). 3H-palmitic acid added at the luminal side of isolated midguts of R. prolixus females was readily absorbed and was used to synthesise phospholipids (80%) and NL (20%). The highest incorporation of 3H-palmitic acid was on the first day after a blood meal. The amounts of diacylglycerol (DG) and triacylglycerol synthesised by the tissue decreased in the presence of Lp in the incubation medium. The metabolic fates of 3H-lipids synthesised by the posterior midgut were followed and it was observed that DG was the major lipid released to Lp particles. However, the majority of phospholipids were not transferred to Lp, but remained in the tissue. The phospholipids that were synthesised and accumulated in the posterior midgut were found to be associated with Rhodnius luminal contents as structural components of PMMs.

Interaction of lipophorin with Rhodnius prolixus oocytes: biochemical properties and the importance of blood feeding

Lipophorin (Lp) is the main haemolymphatic lipoprotein in insects and transports lipids between different organs. In adult females, lipophorin delivers lipids to growing oocytes. In this study, the interaction of this lipoprotein with the ovaries of Rhodnius prolixus was characterised using an oocyte membrane preparation and purified radiolabelled Lp (125I-Lp). Lp-specific binding to the oocyte membrane reached equilibrium after 40-60 min and when 125I-Lp was incubated with increasing amounts of membrane protein, corresponding increases in Lp binding were observed. The specific binding of Lp to the membrane preparation was a saturable process, with a Kdof 7.1 ± 0.9 x 10-8M and a maximal binding capacity of 430 ± 40 ng 125I-Lp/µg of membrane protein. The binding was calcium independent and pH sensitive, reaching its maximum at pH 5.2-5.7. Suramin inhibited the binding interaction between Lp and the oocyte membranes, which was completely abolished at 0.5 mM suramin. The oocyte membrane preparation from R. prolixus also showed binding to Lp from Manduca sexta. When Lp was fluorescently labelled and injected into vitellogenic females, the level of Lp-oocyte binding was much higher in females that were fed whole blood than in those fed blood plasma.

Rhodnius prolixus Malpighian tubules and control of diuresis by neurohormones

Rhodnius prolixus Malpighian tubules (MTs) are a good model for fluid and ion secretion studies in view of the dramatic postprandial diuresis, which follows its massive blood meals. Ingestion of a blood meal equals to 10-12 times their initial body mass, leads to rapid activation of high output by excretory system, which eliminates 40-50 percent of the fluid mass. Secretion of ions and water is stimulated 1000-fold by serotonin and diuretic hormone. These hormones cooperate synergistically to activate adenylate cyclase activity from MTs cells, which increase the level of intracellular cAMP. The anti-diuretic hormones have also an important role in the fluid maintenance of Rhodnius prolixus. Several hours after insect feeding occurs a reduction in urine flow, that has been thought to result from a decreased diuretic hormone release or from a novel mechanism of anti-diuresis involving insect cardioacceleratory peptide 2b (CAP2b) and cyclic GMP. In this article it is discussed how the hormone regulation of fluid transport is done in Rhodnius prolixus MTs.

Os túbulos de Malpighi (TMs) de Rhodnius prolixus são reconhecidos por serem excelentes modelos para o estudo da secreção de fluidos e íons devido a grande diurese que ocorre quando esses animais se alimentam de sangue. O inseto, após alimentação, pode aumentar seu peso corporal inicial em até 10-12 vezes, o que leva a rápida ativação do sistema excretor, que elimina 40-50 por cento do fluido corporal. A secreção de íons e água é estimulada 1000 vezes pela serotonina e pelos hormônios diuréticos. Esses hormônios agem sinergicamente ativando a adenil ciclase das células dos TMs, aumentando os níveis intracelulares de AMPc. Os hormônios anti-diuréticos também têm um importante papel na manutenção dos fluídos corporais do Rhodnius prolixus. Várias horas após a alimentação do inseto ocorre uma redução do fluxo urinário, o que foi sugerido ser decorrente da diminuição da liberação dos hormônios diuréticos ou da anti-diurese envolvendo o peptídeo cardioaceleratório 2b (CAP2b) e o GMPc. Neste artigo é discutida a regulação hormonal do transporte de fluido nos MTs de Rhodnius prolixus.

Sodium pumps in the Malpighian tubule of Rhodnius sp

Malpighian tubule of Rhodnius sp. express two sodium pumps: the classical ouabain-sensitive (Na+ + K+)ATPase and an ouabain-insensitive, furosemide-sensitive Na+-ATPase. In insects, 5-hydroxitryptamine is a diuretic hormone released during meals. It inhibits the (Na+ + K+)ATPase and Na+ -ATPase activities indicating that these enzymes are involved in fluid secretion. Furthermore, in Rhodnius neglectus, proximal cells of Malpighian tubule exposed to hyperosmotic medium, regulate their volume through a mechanism called regulatory volume increase. This regulatory response involves inhibition of the (Na+ + K+)ATPase activity that could lead to accumulation of active osmotic solute inside the cell, influx of water and return to the normal cell volume. Adenosine, a compound produced in stress conditions, also inhibits the (Na+ + K+)ATPase activity. Taken together these data indicate that (Na+ + K+)ATPase is a target of the regulatory mechanisms of water and ions transport responsible for homeostasis in Rhodnius sp.

Efeito da nutrição sobre o controle neuro-endócrino-imunológico de Rhodnius prolixus

Os componentes da alimentação sangüínea, em adição à distensão abdominal, são importantes para a produção dos ecdisteróides e para estabelecer o processo de ecdise em ninfas de quarto estágio de Rhodnius prolixus. Ninfas de R. prolixus, em jejum, quando inoculadas com Enterobacter cloacae B12, tiveram sua mortalidade relacionada ao período de jejum. Ninfas de R. prolixus alimentadas somente com plasma ou com diferentes componentes do sangue comparadas com insetos alimentados com sangue total tiveram afetada a sua resposta imune: (i) uma redução na atividade de lisozima e peptídeos antibacterianos na hemolinfa quando inoculados com E. cloacae; (ii) uma diminuição no número de hemócitos e na formação de nódulos quando inoculados com bactéria; (iii) uma diminuição da imunidade em insetos alimentados com plasma para destruir a infecção causada pela inoculação de E. cloacae; (iv) o tratamento com alfa-ecdisona reverte a imunodepressão causada pela alimentação de plasma

Entretanto, estes experimentos falharam em mostrar alguma interferência na alimentação com plasma no sistema profenoloxidase, PROpo já que a produção de melanina não foi alterada quando o sistema é estimulado pela presença de bactéria na hemolinfa. Estes resultados são discutidos em relação ao efeito dos componentes da dieta e os hormônios da muda na reação imune dos insetos. Foi estudada também a ativação da PROpo na hemolinfa e corpo gorduroso de adultos de R.prolixus. A fenoloxidase (po) não mostrou significante atividade na hemolinfa de insetos inoculados com meio de cultura estéril, quando incubada com L-dopa. Se os insetos são inoculados com E. cloacae B12 a atividade po tem um considerável aumento. Foi visto que a incubação de hemolinfa fresca com corpo gorduroso homogeneizado em tampão isotônico, E. cloacae e L-dopa, induziu a atividade po in vitro. Os mesmos componentes da reação na ausência de bactéria não foram capazes de induzir a formação de melanina. Entretanto, a incubação de hemolinfa com corpo gorduroso homogeneizado em tampão hipotônico e L-dopa formou melanina, indicando que sob condições hipotônicas a ativação do sistema PROpo não depende da presença de E. cloacae. Análises zimográficas em gel de poliacrilamida (SDS-PAGE) com hemolinfa e corpo gorduroso inoculados com bactéria, e não tratados, demonstraram diferentes resultados na atividade de protease em gelatina. As enzimas foram caracterizadas como metaloproteases ou enzimas ativadas por metal. Experimentos usando a fração solúvel do corpo gorduroso homogeneizado em tampão hipotônico ou corpo gorduroso em tampão isotônico e incubados com E. cloacae in vitro indicaram que esta protease está confinada em células do corpo gorduroso e provavelmente é liberada no meio sob tratamento. A implicação destes achados é discutida em relação à ativação do sistema PROpo na defesa de insetos

Efeito da nutrição sobre o controle neuro-endócrino-imunológico de Rhodnius prolixus / Effect of nutrition on the control neuro-endocrine-immune Rhodnius prolixus

Os componentes da alimentação sangüínea, em adição à distensão abdominal, são importantes para a produção dos ecdisteróides e para estabelecer o processo de ecdise em ninfas de quarto estágio de Rhodnius prolixus. Ninfas de R. prolixus, em jejum, quando inoculadas com Enterobacter cloacae B12, tiveram sua mortalidade relacionada ao período de jejum. Ninfas de R. prolixus alimentadas somente com plasma ou com diferentes componentes do sangue comparadas com insetos alimentados com sangue total tiveram afetada a sua resposta imune: (i) uma redução na atividade de lisozima e peptídeos antibacterianos na hemolinfa quando inoculados com E. cloacae; (ii) uma diminuição no número de hemócitos e na formação de nódulos quando inoculados com bactéria; (iii) uma diminuição da imunidade em insetos alimentados com plasma para destruir a infecção causada pela inoculação de E. cloacae; (iv) o tratamento com alfa-ecdisona reverte a imunodepressão causada pela alimentação de plasma. Entretanto, estes experimentos falharam em mostrar alguma interferência na alimentação com plasma no sistema profenoloxidase, PROpo já que a produção de melanina não foi alterada quando o sistema é estimulado pela presença de bactéria na hemolinfa. Estes resultados são discutidos em relação ao efeito dos componentes da dieta e os hormônios da muda na reação imune dos insetos. Foi estudada também a ativação da PROpo na hemolinfa e corpo gorduroso de adultos de R.prolixus. A fenoloxidase (po) não mostrou significante atividade na hemolinfa de insetos inoculados com meio de cultura estéril, quando incubada com L-dopa. Se os insetos são inoculados com E. cloacae B12 a atividade po tem um considerável aumento. Foi visto que a incubação de hemolinfa fresca com corpo gorduroso homogeneizado em tampão isotônico, E. cloacae e L-dopa, induziu a atividade po in vitro. Os mesmos componentes da reação na ausência de bactéria não foram capazes de induzir a formação de melanina. Entretanto, a incubação de hemolinfa com corpo gorduroso homogeneizado em tampão hipotônico e L-dopa formou melanina, indicando que sob condições hipotônicas a ativação do sistema PROpo não depende da presença de E. cloacae. Análises zimográficas em gel de poliacrilamida (SDS-PAGE) com hemolinfa e corpo gorduroso inoculados com bactéria, e não tratados, demonstraram diferentes resultados na atividade de protease em gelatina. As enzimas foram caracterizadas como metaloproteases ou enzimas ativadas por metal. Experimentos usando a fração solúvel do corpo gorduroso homogeneizado em tampão hipotônico ou corpo gorduroso em tampão isotônico e incubados com E. cloacae in vitro indicaram que esta protease está confinada em células do corpo gorduroso e provavelmente é liberada no meio sob tratamento. A implicação destes achados é discutida em relação à ativação do sistema PROpo na defesa de insetos.