Identification of kinin-related peptides in the disease vector, Rhodnius prolixus

We have used an in silico approach to identify a gene from the blood-gorging vector, Rhodnius prolixus, that is predicted to produce an insect kinin prepropeptide. The prepropeptide is 398 amino acids in length and can potentially produce a large number of kinin-related peptides following post-translational processing. A comparison with other insect kinin precursor sequences demonstrates greatest conservation at the C-terminal region of the kinin peptides. Multiple peptides predicted from the kinin gene are phenotypically expressed in R. prolixus, as revealed by MALDI-TOF MS MS, including 12 kinins and one kinin precursor peptide (KPP). Six of these peptides are characterized by the typical insect kinin C-terminal motif FX₁X₂WGamide and five of these are also found as truncated forms. Five peptides were identified with an atypical, though similar, FX₁X₂WAamide C-terminus. There is also peptide with a C-terminal DDNGamide motif and a number of non-amidated peptides.     

The interaction of feeding and mating in the hormonal control of egg production in Rhodnius prolixus

The evidence relating feeding and mating to hormonal control of egg production in Rhodnius prolixus is reviewed from two perspectives. It identifies crucial areas in which information is lacking, and it attempts to relate the findings, most of which have been obtained on laboratory colonies isolated for many years, to the sylvan life of the insect as an opportunistic micropredator.     

Cloning, expression and processing of the CP2 neuropeptide precursor of Aplysia.

The cDNA sequence encoding the CP2 neuropeptide precursor is identified and encodes a single copy of the neuropeptide that is flanked by appropriate processing sites. The distribution of the CP2 precursor mRNA is described and matches the CP2-like immunoreactivity described previously. Single cell RT-PCR independently confirms the presence of CP2 precursor mRNA in selected neurons. MALDI-TOF MS is used to identify additional peptides derived from the CP2 precursor in neuronal somata and nerves, suggesting that the CP2 precursor may give rise to additional bioactive neuropeptides.     

Biostable insect kinin analogs reduce blood meal and disrupt ecdysis in the blood-gorging Chagas’ disease vector,Rhodnius prolixus

Rhodnius prolixus is a blood-gorging hemipteran that takes blood meals that are approximately 10 times its body weight. This blood meal is crucial for growth and development and is needed to ensure a successful molt into the next instar. Kinins are a multifunctional family of neuropeptides which have been shown to play a role in the control of feeding in a variety of insects. In this study, two biostable Aib-containing kinin analogs were tested to see if they interfere with blood-feeding and subsequent development into the next instar. One of the analogs, 1729 (Ac-R[Aib]FF[Aib]WGa), had no effect on the size of the blood meal or on the subsequent molting of the insect into the next instar. This analog also did not interfere with either short-term or long-term diuresis. The second analog, 1728 ([Aib]FF[Aib]WGa), appeared to be an antifeedant. Insects feeding on blood containing this analog (15 μM) only consumed 60% of the blood meal taken by insects fed on blood without analog. Insects feeding on blood containing 1728 had a slower rate of rapid diuresis (diuresis in the first 3–5 h after feeding) leading to less urine being excreted by 5 days post feeding. The consequence of these effects was that insects fed on 1728 did not molt. This data indicates that the biostable Aib-containing analog 1728 disrupts normal growth and development in the blood-feeding insect, R. prolixus.     

Cloning, expression and processing of the CP2 neuropeptide precursor of Aplysia

The cDNA sequence encoding the CP2 neuropeptide precursor is identified and encodes a single copy of the neuropeptide that is flanked by appropriate processing sites. The distribution of the CP2 precursor mRNA is described and matches the CP2-like immunoreactivity described previously. Single cell RT-PCR independently confirms the presence of CP2 precursor mRNA in selected neurons. MALDI-TOF MS is used to identify additional peptides derived from the CP2 precursor in neuronal somata and nerves, suggesting that the CP2 precursor may give rise to additional bioactive neuropeptides.     

Effect of the brain and corpus cardiacum on egg production in Rhodnius prolixus

Using total egg production corrected for size of blood meal as an index of the activity of the corpus allatum (CA), the effects of various surgical manipulations of the neuroendocrine system have been examined. Isolation of the CA from its nervous connections increases egg production well beyond that of a normal insect, thus confirming that the CA is at least partly controlled by inhibitory nerves from the brain. Removal of the corpora cardiaca (CC) reduces the level of this increased egg production, and decapitation anterior to the CC results in a level of egg production that is greater than that found in females decapitated between the CA and CC. Implanting a CC together with a CA into a decapitated female results in a higher egg production than implanting a CA alone. These results demonstrate that an allatotropic influence is exerted by the CC. Experiments designed to examine the role of the brain were inconclusive and did not eliminate the possibility that the allatotropin from the CC originated in the brain.     

The effect of severing the dorsal vessel on egg production in Rhodnius prolixus

Severing the dorsal vessel (DV) behind the corpus allatum (CA), or in the anterior part of the abdomen of Rhodnius prolixus, greatly reduces egg production, an effect which is abolished by the topical application of juvenile hormone l (JH l). Severing the DV in the posterior abdomen does not result in a marked reduction of egg production, although severing the alary muscles in segments V and VI has a similar effect to severing the DV in the anterior abdomen. Reduced egg production caused by severing the DV on day 8 postemergence does not occur if the nerves connecting the CA to the brain are severed on day 1 post emergence. However, egg production is reduced if the DV is severed on day 1 post emergence and the connections between the brain and the CA severed on day 8, suggesting that inhibition of the CA caused by severance of the DV requires innervation from the brain. An isolated CA implanted into an animal decapitated immediately after feeding escapes from the inhibition imposed by severance of the DV. Conversely, the CA in an insect, the head of which has been decapitated just anterior to the CA, remains inhibited. This result suggests that the head posterior to the brain must be present to maintain inhibition. It is concluded that DV severance acts on the brain via some humoral influence to impose inhibition on the CA, and that an endocrine center in the head is required in order to maintain the inhibition.     

Partial localization of a brain factor inhibiting egg production in the blood-feeding insect,Rhodnius prolixus

A previous study by Davey [Can J Zool 4:243–249 (1987)] showed that egg production in the blood-feeding insect Rhodnius prolixus is greatly enhanced when the corpus allatum (CA) is denervated. This result supported findings of others that the brain of Rhodnius imposes an inhibition on the CA via its connections to the CA. The present study identifies the nervus corporis cardiacum II (NCCII) as the nerve responsible for this inhibitory influence. Transecting the NCCII before feeding causes a dramatic increase in the number of eggs made. Since the NCCII is a relatively small nerve that may contain only axons of lateral and posterior neurosecretory cells in the protocerebrum, this result suggests that one or both of these cell types produce the brain factor inhibiting egg production in this insect. Arch. Insect Biochem. Physiol. 39:126–131, 1998. © 1998 Wiley-Liss, Inc.     

Silencing the odorant receptor co-receptor RproOrco affects the physiology and behavior of the Chagas disease vector Rhodnius prolixus

Olfaction is one of the main sensory modalities that allow insects to interpret their environment. Several proteins, including odorant-binding proteins (OBPs) and odorant receptors (ORs), are involved in this process. Odorant receptors are ion channels formed by a binding unit OR and an odorant receptor co-receptor (Orco). The main goal of this study was to characterize the Orco gene of Rhodnius prolixus (RproOrco) and to infer its biological functions using gene silencing. The full-length RproOrco gene sequence was downloaded from VectorBase. This gene has 7 introns and is located in the genome SuperContig GL563069: 1,017,713–1,023,165. RproOrco encodes a protein of 473 amino acids, with predicted 7 transmembrane domains, and is highly expressed in the antennae during all R. prolixus developmental stages. The RNAi technique effectively silenced RproOrco, reducing the gene's expression by approximately 73%. Interestingly, the effect of gene silencing persisted for more than 100 days, indicating a prolonged effect of dsRNA that was maintained even after molting. The phenotypic effects of silencing involved the following: (1) loss of the ability to find a vertebrate host in a timely manner, (2) decreased ingested blood volume, (3) delayed and decreased molt rate, (4) increased mortality rate, and (5) decreased egg laying. Our data strongly suggest that dsOrco disrupts R. prolixus host-finding behavior, which is further reflected in the blood ingestion, molting, mortality, and egg laying data. This study clearly demonstrates that Orco is an excellent target for controlling triatomine populations. Thus, the data presented here open new possibilities for the control of vector-borne diseases.     

Zebrafish genes for neuropeptide Y and peptide YY reveal origin by chromosome duplication from an ancestral gene linked to the homeobox cluster

Neuropeptide Y (NPY) and peptide YY (PYY) are related 36-amino acid peptides. NPY is widely distributed in the nervous system and has several physiological roles. PYY serves as an intestinal hormone as well as a neuropeptide. We report here cloning of the npy and pyy genes in zebrafish (Danio rerio). NPY differs at only one to four amino acid positions from NPY in other jawed vertebrates. Zebrafish PYY differs at three positions from PYY from other fishes and at 10 positions from mammals. In situ hybridization showed that neurons containing NPY mRNA have a widespread distribution in the brain, particularly in the telencephalon, optic tectum, and rhombencephalon. PYY mRNA was found mainly in brainstem neurons, as reported previously for vertebrates as divergent as the rat and the lamprey, suggesting an essential role for PYY in these neurons. PYY mRNA was observed also in the telencephalon. These results were confirmed by immunocytochemistry. As in the human, the npy gene is located adjacent to homeobox (hox) gene cluster A (copy a in zebrafish), whereas the pyy gene is located close to hoxBa. This suggests that npy and pyy arose from a common ancestral gene in a chromosomal duplication event that also involved the hox gene clusters. As zebrafish has seven hox clusters, it is possible that additional NPY family genes exist or have existed. Also, the NPY receptor system seems to be more complex in zebrafish than in mammals, with at least two receptor genes without known mammalian orthologues.     

The distribution and effects of Dippu-allatostatin-like peptides in the blood-feeding bug, Rhodnius prolixus

Using a polyclonal antiserum to Dippu-allatostatin 7 (Dippu-AST 7; formerly AST 1) of the cockroach Diploptera punctata, we have demonstrated the presence of AST-like immunoreactivity (ALI) in cells and processes throughout the nervous system, gut, and peripheral tissues of unfed fifth instar and adult Rhodnius prolixus. ALI in apparent neurosecretory cells of the brain, suboesophageal ganglion, and mesothoracic ganglionic mass, as well as in midgut endocrine cells, suggests that Rhodnius allatostatins may act as neurohormones/hormones. The presence of ALI in possible interneurons and areas of neuropile throughout the CNS also suggests roles as neuromodulators and/or neurotransmitters. Dippu-AST 7 inhibits spontaneous and leucokinin 1 (LK 1)-induced contractions of the Rhodnius hindgut in a dose-dependent manner. The low concentrations capable of inhibiting both spontaneous (10(-12)M) and LK 1-induced contractions (10(-10) to 10(-9)M) suggest that ASTs may be acting as neurohormones/hormones on the hindgut. We have also shown that Dippu-AST 7 influences the muscle activity of the Rhodnius dorsal vessel at concentrations as low as 10(-11)M.