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.

Corticotropin-releasing factor-like diuretic hormone acts as a gonad-inhibiting hormone in adult female, Rhodnius prolixus.

Within insects, corticotropin-releasing factor/diuretic hormones (CRF/DHs) are responsible for the modulation of a range of physiological and behavioural processes such as feeding, diuresis, and reproduction. Rhopr-CRF/DH plays a key role in feeding and diuresis in Rhodnius prolixus, a blood-gorging insect and a vector for human Chagas disease. Here, we extend our understanding on the role of this neurohormone in reproduction in adult female R. prolixus. Double-label immunohistochemistry displays co-localized staining of CRF-like and the glycoprotein hormone (GPA2/GPB5) subunit GPB5-like immunoreactivity in the same neurosecretory cells (NSCs) in the mesothoracic ganglionic mass (MTGM) and in their neurohemal sites in adult female R. prolixus, suggesting these peptides could work together to regulate physiological processes. qPCR analysis reveals that the transcript for Rhopr-CRF/DH receptor 2 (Rhopr-CRF/DH-R2) is expressed in reproductive tissues and fat body (FB) in adult female R. prolixus, and its expression increases post blood meal (PBM), a stimulus that triggers diuresis and reproduction. Using RNA interference, transcript expression of Rhopr-CRF/DH-R2 was knocked down, and egg production monitored by examining the major yolk protein, vitellogenin (Vg), the number and quality of eggs laid, and their hatching ratio. Injection of dsCRFR2 into adult females reduces Rhopr-CRF/DH-R2 transcript expression, accelerates oogenesis, increases the number of eggs produced, and reduces hatching rate in female R. prolixus. Downregulation of Rhopr-CRF/DH-R2 leads to an increase in the transcript expression of RhoprVg1 in the fat body and ovaries, and increases the transcript level for the Vg receptor, RhoprVgR, in the ovaries. A significant increase in Vg content in the fat body and in the hemolymph is also observed. Incubation of isolated tissues with Rhopr-CRF/DH leads to a significant decrease in transcript expression of RhoprVg1 in the fat body and RhoprVg1 in the ovaries. In addition, Rhopr-CRF/DH reduces transcript expression of the ecdysteroid biosynthetic enzymes and reduces ecdysteroid titer in the culture medium containing isolated ovaries. These results suggest the involvement of the CRF-signaling pathway in reproduction, and that Rhopr-CRF/DH acts as a gonad-inhibiting hormone in the adult female R. prolixus, as previously shown for the colocalized glycoprotein, GPA2/GPB5.

Malpighian tubules of Rhodnius prolixus: More than post-prandial diuresis.

Rhodnius prolixus, a major vector of Chagas disease, may be considered the model upon which the foundations of insect physiology and biochemistry were built. It is an obligate blood feeder in which the blood meal triggers growth, development and reproduction. The blood meal also triggers a post-prandial diuresis to maintain osmotic homeostasis. In R. prolixus, as with other insects, the Malpighian tubules play a critical role in this diuresis, and much has been learned about diuresis in R. prolixus, and in other model insects. But the post-genomic era has brought new insights, identifying functions quite apart from diuresis for Malpighian tubules. Indeed, microarrays, transcriptomes, and proteomics have revealed the major roles that Malpighian tubules play in immunity, detoxification, pesticide resistance, and in tolerance to overall stress. This is particularly relevant to R. prolixus since gorging on blood creates several challenges in addition to osmotic balance. Xenobiotics may be present in the blood or toxins may be produced by metabolism of blood; and these must be neutralized and excreted. These processes have not been well described at the molecular level for Malpighian tubules of R. prolixus. This paper will review the involvement of Malpighian tubules in immunity and detoxification, identifying new aspects for Malpighian tubule physiology of R. prolixus by virtue of a transcriptome analysis. The transcriptome analysis indicates the potential of Malpighian tubules of R. prolixus to mount a robust innate immune response, and to contribute to antioxidant production and heme detoxification.

Exploring the role of glycoprotein hormone GPA2/GPB5 in the medically important insect, Rhodnius prolixus.

Glycoprotein hormones are formed by the heterodimerization of alpha and beta subunits. In vertebrates, there are five glycoprotein hormones, four of which have a common alpha subunit (GPA1) bound to a specific beta subunit (GPB1, GPB2, GPB3, or GPB4), and the fifth, thyrostimulin, is formed by the dimerization of GPA2 and GPB5 subunits. These hormones mediate physiological events such as development, metabolism, and reproduction, although the functional role of thyrostimulin in vertebrates has not been fully elucidated. Recent reports in invertebrates, specifically in holometabolous insects, suggest that GPA2/GPB5 plays a critical role in development, diuresis, and reproduction. In this study, we clone and characterize the transcripts for the glycoprotein hormone GPA2/GPB5 and its receptor (LGR1) in fifth instar Rhodnius prolixus, a hemimetabolous insect vector of Chagas disease. Sequence analyses reveals considerable identity and similarity between GPA2/GPB5 and LGR1 and those reported in other arthropod species. Quantitative PCR (qPCR) shows that both subunit transcripts, GPA2 and GPB5, and LGR1 transcripts are present in a variety of tissues, with greatest expression of the subunits in the central nervous system (CNS) and highest LGR1 expression in the Malpighian tubules (MT). Results from temporal qPCR analyses reveal a decrease in transcript expression 24 h after feeding, followed by an increase as the days post-feeding advance. Using immunohistochemistry, we show that GPB5 is expressed throughout the CNS, and importantly is present in neurosecretory cells in the brain and abdominal neuromeres and their neurohemal organs, indicating a neurohormonal role for this signaling pathway. A reduction in LGR1 transcript expression (via RNA interference) led to a greater weight loss and mortality rate in unfed insects. In addition, when a blood meal is offered, the insects with reduced LGR1 consume a significantly smaller blood meal and have higher mortality rates as the days post-feeding advance. Overall, the results suggest that the GPA2/GPB5 signaling pathway may play roles during a prolonged unfed state and in feeding-related events.

Investigating the role of glycoprotein hormone GPA2/GPB5 signaling in reproduction in adult female Rhodnius prolixus.

Glycoprotein hormones are essential for regulating various physiological activities in vertebrates and invertebrates. In vertebrates, the classical glycoprotein hormones include follicle-stimulating hormone (FSH), luteinizing hormone (LH), thyroid-stimulating hormone (TSH) and chorionic gonadotropin (CG), which have crucial roles in growth, development, metabolism, and reproduction. In female mammals, FSH stimulates egg production in the ovaries, whereas LH and CG act as the triggers for follicular ovulation. The more recently discovered heterodimeric glycoprotein hormone GPA2/GPB5 (called thyrostimulin in vertebrates) is suggested to be involved in reproductive processes in arthropods. Here, we focus on understanding the role of GPA2/GPB5 and its receptor, LGR1, in the reproductive success of adult female Rhodnius prolixus, a vector of Chagas disease. qPCR was used to monitor the expression of GPA2 and GPB5 transcripts and their receptor in different tissues. Immunohistochemistry was used to show the distribution of GPB5 in the nervous system and reproductive system, and RNA interference was used to disrupt the glycoprotein hormone signaling pathway. Both subunit transcripts, GPA2 and GPB5, are present in a variety of tissues, with the greatest expression in the central nervous system; whereas the LGR1 transcript is present in peripheral tissues, including the fat body and the reproductive system of adult females. In the adult female, GPB5-like immunoreactive axonal projections are present in the trunk nerves extending onto the reproductive tissues, with processes overlaying the ovaries, oviducts, spermatheca, and bursa, indicating the possibility of neural control by neurons containing GPA2/GPB5. In addition, GPB5-like immunostaining is present in muscles encircling the ovarioles, and in the cytoplasm of trophocytes (nurse cells) located in the tropharium. GPB5-like immunoreactive processes and blebs are also localized to the previtellogenic follicles, suggesting an involvement of this glycoprotein hormone signaling in oocyte development. LGR1 transcript expression increases in the adult female reproductive system post-feeding, a stimulus that initiates reproductive development, adding further support to an involvement in reproduction. We have investigated the effect of LGR1 downregulation on reproductive processes, monitoring the number and the quality of eggs laid, hatching ratio, and production of vitellogenin (Vg), the major yolk protein for developing eggs. Downregulation of LGR1 leads to increases in transcript expression of vitellogenin, RhoprVg1, in the fat body and the vitellogenin receptor, RhoprVgR, in the ovaries. Total protein in the fat body and hemolymph of dsLGR1-injected insects increased compared to controls and associated with this effect was a significant increase in vitellogenin in these tissues. dsLGR1-injection leads to accelerated oogenesis, an increase in the number of eggs produced and laid, an increase in egg size and a reduction in hatching rate. Our results indicate that GPA2/GPB5 signaling acts to delay egg production in adult female R. prolixus.