Characterization and expression of a long neuropeptide F (NPF) receptor in the Chagas disease vector Rhodnius prolixus.

In this study, a long neuropeptide F receptor of the blood-feeding hemipteran, Rhodnius prolixus (RhoprNPFR) has been cloned and characterized. Approximately 70% of the RhoprNPFR deduced protein sequence is identical to that of other hemipteran NPFRs. RhoprNPFR has seven highly-conserved transmembrane domains, two cysteine residues in the 2nd and 3rd extracellular loops that likely form a disulfide bond integral for maintaining the structure of the receptor, and a conserved DRY motif after the third transmembrane domain. All of these characteristics are typical of class A rhodopsin-like GPCRs. The receptor transcript is predominantly expressed in the central nervous system (CNS) and gut of both fifth instar and adult R. prolixus. Using fluorescent in situ hybridization (FISH), we identified six bilaterally-paired large median neurosecretory cells (approximately 30µm in diameter) in the brain that express the RhoprNPFR mRNA transcript. We also found RhoprNPFR transcript expression in endocrine cells in the anterior midgut of fifth instars, as well as in putative pre-follicular cells present in the germarium and between developing oocytes, and in the nutritive cord. These results suggest that RhoprNPFR may play a role within the CNS, and in digestion and possibly egg production and/or egg development in R. prolixus.

Cloning and expression of long neuropeptide F and the role of FMRFamide-like peptides in regulating egg production in the Chagas vector, Rhodnius prolixus.

Long neuropeptide F (NPF) is a neuropeptide implicated in the control of feeding, digestion and reproduction in various insect species. Here we have isolated the cDNA sequence encoding NPF in Rhodnius prolixus (RhoprNPF). The RhoprNPF gene is composed of 3 exons and 2 introns, one of which is present in the peptide coding region. RhoprNPF is 42 amino acids long and has the characteristic RFamide C-terminus, which is common of FMRFamide-like peptides (FLPs). Quantitative PCR (qPCR) shows that RhoprNPF mRNA is present in higher amounts in fifth instars than in adults, implying that it may play a role in growth and development. In situ hybridization shows that the RhoprNPF transcript is present in median neurosecretory cells (MNSCs) in the brain, cells in the fifth instar hindgut and cells along the longitudinal muscle fibers of the adult female lateral oviducts. Injection of the last 8 amino acids of RhoprNPF (truncated RhoprNPF, AVAGRPRFa), which is considered to be the active core sequence for biological activity, into mated, fed, female adult R. prolixus decreased the number of eggs found in the ovaries as well as increased the number of eggs laid. This suggests that RhoprNPF may play a role in accelerating the process of ovulation from the ovary of the female R. prolixus. An increase in oogenesis was observed following the injection of other FLPs such as RhoprShortNPF, GNDNFMRFamide and AKDNFIRFamide, whereas the FLP, RhoprMS, and the allatostatin, RhoprAST-2, inhibited egg production.

Functional characterization and expression analysis of the myoinhibiting peptide receptor in the Chagas disease vector, Rhodnius prolixus.

Myoinhibiting peptides (MIPs), which are also known as B-type allatostatins, are a family of neuropeptides found in protostomes. Their primary structure is characterized by an amidated carboxyl-terminal motif consisting of a conserved pair of tryptophan residues normally separated by six non-conserved amino acids (W(X6)Wamide). In the fruit fly Drosophila melanogaster, MIPs are likely the ancestral ligands of the sex peptide receptor, which plays an important role in courtship and reproduction. Recently, several endogenous MIPs were discovered in the Chagas disease vector, Rhodnius prolixus, having both conserved (W(X6)Wamide) and atypical (W(X7)Wamide) carboxyl-terminal motifs. Physiological functions of MIPs are plentiful and include inhibition of visceral muscle activity; a role that has been illustrated on hindgut in R. prolixus. In order to identify novel physiological targets and elucidate biological actions for the MIPs in R. prolixus, we have isolated and examined the spatial expression profile of the MIP receptor transcript in various fifth instar tissues and have additionally determined the expression profile in reproductive tissues of fifth instar as well as adult insects. The most abundant MIP receptor transcript expression was found in the salivary glands and central nervous system, which corroborates roles previously determined for MIPs in other insects. We functionally-characterized the endogenous MIP receptor and examined its activation by R. prolixus MIPs containing the typical W(X6)Wamide and atypical W(X7)Wamide carboxyl-terminal motifs. These peptides dose-dependently activated the MIP receptor (RhoprMIPr1) with EC50 values in the mid-nanomolar range. We also examined the activity of these RhoprMIPs on spontaneous muscle contractions of oviducts from female adult R. prolixus. Our findings confirm the myoinhibitory nature of the MIP peptides, which dose-dependently reduced spontaneous oviduct contractions by nearly 70%, again having mid-nanomolar EC50 values. Finally, we utilized a heterologous receptor assay and oviduct bioassay to examine the activity of several MIP structural analogs, which independently confirmed the requirement of the highly conserved tryptophan residues as well as the amidated C-terminus for retaining full biological activity.

Myoinhibitors controlling oviduct contraction within the female blood-gorging insect, Rhodnius prolixus.

Muscle activity can be regulated by stimulatory and inhibitory neuropeptides allowing for contraction and relaxation. There are various families of neuropeptides that can be classified as inhibitors of insect muscle contraction. This study focuses on Rhodnius prolixus and three neuropeptide families that have been shown to be myoinhibitors in insects: A-type allatostatins, myoinhibiting peptides (B-type allatostatins) and myosuppressins. FGLa/AST-like immunoreactive axons and blebs were found on the anterior of the dorsal vessel and on the abdominal nerves. FGLa/AST-like immunoreactive axons were also seen in the trunk nerves and on the bursa. The effects of RhoprAST-2 (FGLa/AST or A-type allatostatins) and RhoprMIP-4 (MIP/AST or B-type allatostatins) were similar, producing dose-dependent inhibition of R. prolixus spontaneous oviduct contractions with a maximum of 70% inhibition and an EC50 at approximately 10(-8)M. The myosuppressin of R. prolixus (RhoprMS) has an unusual FMRFamide C-terminal motif (pQDIDHVFMRFa) as compared to myosuppressins from other insects. Quantitative PCR results show that the RhoprMS receptor transcript is present in adult female oviducts; however, RhoprMS does not have an inhibitory effect on R. prolixus oviduct contractions, but does have a dose-dependent inhibitory effect on the spontaneous contraction of Locusta migratoria oviducts. SchistoFLRFamide, the myosuppressin of Schistocerca gregaria and L. migratoria, also does not inhibit R. prolixus oviduct contractions. This implies that FGLa/ASTs and MIP/ASTs may play a role in regulating egg movement within the oviducts, and that the myosuppressin although myoinhibitory on other muscles in R. prolixus, does not inhibit the contractions of R. prolixus oviducts and may play another role in the reproductive system.

The female reproductive system of the kissing bug, Rhodnius prolixus: arrangements of muscles, distribution and myoactivity of two endogenous FMRFamide-like peptides.

Phalloidin staining F-actin was used to image muscle fiber arrangements present in the reproductive system of the adult female Rhodnius prolixus. A mesh of muscle fibers encircles the ovaries whereas a criss-cross pattern of finer muscle fibers covers each ovariole. Two layers of muscle fibers (arranged longitudinally and circularly) form the lateral oviducts. The circular layer of muscle fibers extends throughout the common oviduct and spermathecae. A chevron pattern of thicker muscle fibers makes up the bursa. All of these structures show spontaneous contractions that are stimulated in a dose-dependent manner by the endogenous peptides, GNDNFMRFamide and AKDNFIRFamide which belong to the family of the FMRFamide-like peptides (FLP). Immunohistochemistry shows that these peptides could be supplied via nerves to the oviducts, spermathecae and bursa. Although no FMRF-like immunoreactivity was observed on the ovarioles/ovary they still exhibited a stimulatory response to the peptides indicating that they may be under the influence of FLPs as neurohormones. This work implicates FLPs in the control of ovulation, egg movement and oviposition in this insect.