Jean-Paul Paluzzi

Distribution, activity and evidence for the release of an anti-diuretic peptide in the kissing bug Rhodnius prolixus.

SUMMARY In the haematophagous insect Rhodnius prolixus, diuresis is accomplished through the combined actions of peptidergic diuretic hormones and 5-HT released from neurohaemal sites on the abdominal nerves. Preliminary work on anti-diuresis in this blood-feeder, previously believed to occur through a decrease in the levels of the diuretic factors, indicates that an anti-diuretic hormone, with properties similar to CAP2b (pELYAFPRVamide; recently renamed Mas-CAPA-1), might also be present in R. prolixus. Here, we present evidence from immunohistochemical analysis that suggests a PRXamide-like neuropeptide may be released from the abdominal neurohaemal sites beginning 3–4 h following feeding; a time that coincides with the cessation of diuresis. We also show evidence for an endogenous factor, isolated from the central nervous system using reversed-phase high performance liquid chromatography, which mimics the effects of Mas-CAPA-1. Specifically, this endogenous anti-diuretic factor inhibits rates of 5-HT-stimulated secretion in a dose-dependent manner and elevates intracellular cGMP levels of Malpighian tubules stimulated with 5-HT.

Isolation, expression analysis, and functional characterization of the first antidiuretic hormone receptor in insects

Diuresis following blood-gorging in Rhodnius prolixus is the major process leading to the transmission of Chagas’ disease. We have cloned the cDNA of the first receptor known to be involved in an antidiuretic strategy in insects, a strategy that prevents diuresis. This receptor belongs to the insect CAPA receptor family known in other insects to be activated by peptides encoded within the capability gene. We characterize the expression profile in fifth-instars and find expression is localized to the alimentary canal. Highest transcript levels are found in Malpighian tubules and the anterior midgut, which are known targets of the antidiuretic hormone, RhoprCAPA-α2. Two transcripts were identified, capa-r1 and capa-r2; however, the latter encodes an atypical G protein-coupled receptor lacking a region ranging between the first and second transmembrane domain. Our heterologous expression assay revealed the expressed capa-r1 receptor is activated by RhoprCAPA-α2 (EC50 = 385nM) but not by RhoprCAPA-α1. Structural analogs of the inactive RhoprCAPA-α1 were capable of activating the expressed capa-r1 receptor, confirming the importance of the C-terminal consensus sequence common to CAPA-related peptides. In addition, this receptor has some sensitivity to the pyrokinin-related peptide, RhoprCAPA-αPK1, but with an efficacy ≈40-fold less than RhoprCAPA-α2. Other peptides belonging to the PRXamide superfamily were inactive on the capa-r1 receptor. Taken together, the neuroendocrinological relevance of this receptor in facilitating the antidiuretic strategy in R. prolixus may make this receptor a useful target for development of agonists or antagonists that could help influence the transmission of Chagas’ disease that occurs during diuresis in this medically important insect-disease vector.

A second gene encodes the anti-diuretic hormone in the insect, Rhodnius prolixus

In the haematophagous insect, Rhodnius prolixus, a rapid diuresis following engorgement of vertebrate blood is under the control of two main diuretic hormones: a corticotropin-releasing factor (CRF)-related peptide andserotonin (5HT). A CAP2b (CAPA)-related peptide is involved in the termination of this diuresis, and we have recently identified a gene, now referred to as RhoprCAPA-alpha, encoding CAPA peptides in R. prolixus. Here we identify a second gene, RhoprCAPA-beta, which also encodes CAPA peptides and characterize its expression in fifth-instar and adults. The RhoprCAPA-beta gene is more highly expressed in the CNS than the RhoprCAPA-alpha gene, but neither gene is expressed in other tested adult tissues. Both genes are expressed in a subset of immunoreactive neurons identified using an antisera which recognizes CAP2b-related peptides. The expression of each paralog is modified by feeding and we propose this to be a result of requirements of anti-diuretic regulation during salt and water homeostasis.

Identification, spatial expression analysis and functional characterization of a pyrokinin-1 receptor in the Chagas' disease vector, Rhodnius prolixus.

The capability or capa gene, encodes a pyrokinin-related peptide (known as pyrokinin-1, PK1) that contains the consensus carboxy-terminal sequence of WFGPRL-NH(2). Although the CAPA precursor polypeptide in Rhodnius prolixus yields the anti-diuretic hormone, RhoprCAPA-α2, no function has yet been elucidated for the pyrokinin-1 peptide, RhoprCAPA-αPK1. In order to elucidate the possible physiological roles of the PK1-related peptides in R. prolixus, we have isolated and functionally characterized the PK1 receptor, RhoprPK1-R. Additionally, we have determined a set of three optimal reference genes to utilize for normalization of data obtained when carrying out spatial expression analyses via quantitative reverse transcriptase PCR (RT-qPCR) in various tissues of fifth instar R. prolixus. The RhoprPK1-R expression profile differs strikingly from the receptor for the anti-diuretic hormone RhoprCAPA-α2, which is localized mainly to gut epithelial tissues. Instead, RhoprPK1-R expression in fifth instar stage insects was identified in tissues that are not involved in osmotic and ionic balance, including the prothoracic glands, male reproductive tissues and a pooled sample composed of fat body, dorsal vessel, abdominal nerves and female reproductive tissues. Thus, this research establishes novel possibilities for the physiological roles of the pyrokinin-related peptides in this medically relevant disease vector.

Identification of the elusive peptidergic diuretic hormone in the blood-feeding bug Rhodnius prolixus : a CRF-related peptide

SUMMARY Probing of a host and ingestion of a blood-meal in a fifth instar Rhodnius prolixus results in a cascade of tightly integrated events. The huge blood-meal is pumped into the anterior midgut during feeding, then modified by diuresis and stored until it is digested. While serotonin is known to be a diuretic hormone in R. prolixus, a peptidergic factor(s) was also known to play a role in diuresis. In the present study we employed molecular techniques and mass spectrometry to determine the sequence of a native CRF-like peptide from R. prolixus (Rhopr DH). In addition, we confirmed the distribution and localization of Rhopr DH using in situ hybridization and immunohistochemistry, and demonstrated its potent biological activity on both the anterior midgut and Malpighian tubules.

The Heterodimeric Glycoprotein Hormone, GPA2/GPB5, Regulates Ion Transport across the Hindgut of the Adult Mosquito, Aedes aegypti

A family of evolutionarily old hormones is the glycoprotein cysteine knot-forming heterodimers consisting of alpha- (GPA) and beta-subunits (GPB), which assemble by noncovalent bonds. In mammals, a common glycoprotein hormone alpha-subunit (GPA1) pairs with unique beta-subunits that establish receptor specificity, forming thyroid stimulating hormone (GPA1/TSHβ) and the gonadotropins luteinizing hormone (GPA1/LHβ), follicle stimulating hormone (GPA1/FSHβ), choriogonadotropin (GPA1/CGβ). A novel glycoprotein heterodimer was identified in vertebrates by genome analysis, called thyrostimulin, composed of two novel subunits, GPA2 and GPB5, and homologs occur in arthropods, nematodes and cnidarians, implying that this neurohormone system existed prior to the emergence of bilateral metazoans. In order to discern possible physiological roles of this hormonal signaling system in mosquitoes, we have isolated the glycoprotein hormone genes producing the alpha- and beta-subunits (AedaeGPA2 and AedaeGPB5) and assessed their temporal expression profiles in the yellow and dengue-fever vector, Aedes aegypti. We have also isolated a putative receptor for this novel mosquito hormone, AedaeLGR1, which contains features conserved with other glycoprotein leucine-rich repeating containing G protein-coupled receptors. AedaeLGR1 is expressed in tissues of the alimentary canal such as the midgut, Malpighian tubules and hindgut, suggesting that this novel mosquito glycoprotein hormone may regulate ionic and osmotic balance. Focusing on the hindgut in adult stage A. aegypti, where AedaeLGR1 was highly enriched, we utilized the Scanning Ion-selective Electrode Technique (SIET) to determine if AedaeGPA2/GPB5 modulated cation transport across this epithelial tissue. Our results suggest that AedaeGPA2/GPB5 does indeed participate in ionic and osmotic balance, since it appears to inhibit natriuresis and promote kaliuresis. Taken together, our findings imply this hormone may play an important role in ionic balance when levels of Na+ are limited and levels of K+ are in excess – such as during the digestion and assimilation of erythrocytes following vertebrate blood-feeding by females.

The antidiuretic neurohormone RhoprCAPA-2 downregulates fluid transport across the anterior midgut in the blood-feeding insect Rhodnius prolixus

Osmotic balance in insects is regulated by the excretory system, consisting of Malpighian tubules and the gut under the control of diuretic and antidiuretic factors. Terrestrial insects must conserve water, and antidiuresis is the norm, only interrupted by brief diuretic periods. Surprisingly, little is known about antidiuresis in insects. Two antidiuretic strategies have been described. The first antidiuretic mechanism involves the reabsorption of fluid from the primary urine in the hindgut. More recently, a second antidiuretic strategy was reported, consisting of inhibition of primary urine formation by the Malpighian tubules. Recently, we isolated, characterized, and cloned the gene encoding for the antidiuretic neurohormone (the neuropeptide RhoprCAPA-2) acting on the Malpighian tubules of Rhodnius prolixus. Here we describe a third, novel mechanism central to the antidiuretic strategy of R. prolixus, the inhibition of ion and fluid transport across the anterior midgut by RhoprCAPA-2. Our results show that RhoprCAPA-2 (1 micromol/l) reduces serotonin-stimulated fluid transport from 83 +/- 11 to 12 +/- 12 nl/min and equivalent short-circuit current from 20 +/- 4 to 5 +/- 0.7 microA/cm(2) in diuretic hormone-stimulated anterior midgut. RhoprCAPA-2 appears to function independently of intracellular cGMP or Ca(2+) in the midgut. Thus, the antidiuretic neurohormone RhoprCAPA-2 has multiple target tissues, and we hypothesize that RhoprCAPA-2 functions to coordinate the transport activity of the anterior midgut and Malpighian tubules so that the rate of fluid transport into the haemolymph by the anterior midgut matches the transport rate of Malpighian tubules to maintain the volume and ion composition of haemolymph.

Isolation and characterization of the cDNA encoding DH31 in the kissing bug, Rhodnius prolixus

Rhodnius prolixus undergoes a period of rapid diuresis after ingesting large blood meals. Neurohormones with either diuretic or anti-diuretic activity control diuresis by acting on several tissues including the Malpighian tubules. One of the neurohormones that potentially plays a role in diuresis is diuretic hormone 31 (DH(31)) which belongs to the insect calcitonin-like family of diuretic hormones. Here we determine the complete cDNA sequences of three Rhopr-DH(31) splice variants (Rhopr-DH(31)-A, Rhopr-DH(31)-B and Rhopr-DH(31)-C) and characterize their expression in unfed fifth-instar R. prolixus. Reverse transcriptase-PCR demonstrates that Rhopr-DH(31) is predominantly expressed in the central nervous system (CNS) of unfed fifth-instars. However, the expression of the three splice variants differs with Rhopr-DH(31)-B expression being the highest followed by Rhopr-DH(31)-A and Rhopr-DH(31)-C, as determined using semi-quantitative Southern blot analysis. Fluorescent in situ hybridization reveals that Rhopr-DH(31) is expressed in a variety of cells in the CNS, including some neurosecretory cells.

Expression analysis and molecular characterization of aquaporins in Rhodnius prolixus

Aquaporins (AQPs) are water channels responsible for transport of water and, in some cases, transport of small solutes such as urea and glycerol across lipid bilayer membranes. Hematophagous insects, such as Rhodnius prolixus, ingest large volumes of fluid and must rapidly eliminate the excess of water and salts from the blood meal within the gut. In order to deal with this increase in body fluid volume, a hormone-controlled diuresis is activated, during which a high rate of water and salt absorption occurs across the anterior midgut, followed by secretion of water and salts by the Malpighian tubules (MTs). Previously, one member of the MIP family (major intrinsic protein that includes the AQP family) was identified in the MTs of R. prolixus, and named RpMIP. We have described here that the RpMIP gene has different variants, and is present in tissues other than MTs. In addition, we have characterized a new AQP (RhoprAQP1) found in different tissues of R. prolixus. The expression of these transcripts in unfed insects as well as blood fed insects was evaluated using real-time quantitative PCR. Molecular models of the predicted proteins were constructed and the characteristics of their pores evaluated. A yeast complementation assay was used to validate that the products of these transcripts were bona fide AQPs. Both RhoprAQP1 and RhoprMIP-A were capable of transporting water whereas RhoprMIP-A was also capable of transporting H2O2. Taken together, these analyses suggest that RhoprMIP is probably an aquaglyceroporin, while RhoprAQP1 appears to be a strict aquaporin that transports only water.

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.