The glycoprotein hormone receptor (LGR1) influences Malpighian tubule secretion rate in Rhodnius prolixus.

In the hemipteran Rhodnius prolixus, successful post-prandial diuresis is accomplished through the synergistic actions of the peptidergic diuretic hormone RhoprCRF/DH and the biogenic amine 5-hydroxytryptamine (5-HT), and by an antidiuretic hormone RhoprCAPA-2 that terminates diuresis by inhibiting this synergy. Lateral neurosecretory cells (NSCs) in the mesothoracic ganglionic mass release RhoprCRF/DH, while midline NSCs release RhoprCAPA-2 during blood feeding. These NSCs co-express GPA2/GPB5, a conserved glycoprotein hormone involved in various physiological processes across bilaterians. This study investigates the influence of GPA2/GPB5 signaling on Malpighian tubule (MT) fluid secretion in R. prolixus. GPB5-like immunoreactivity in lateral and midline NSCs decreases following a blood meal, suggesting release and a role in diuresis. Downregulating the GPA2/GPB5 receptor LGR1 via RNA interference results in an increased basal fluid secretion rate in MTs, which is inhibited by the antidiuretic hormone RhoprCAPA-2. dsLGR1 treatment reduces the effects of RhoprCRF/DH and 5-HT on MT secretion and eliminates their synergism. RT-qPCR reveals that the transcript expression of the diuretic and antidiuretic hormone receptors are decreased in MTs of dsLGR1 injected insects, indicating that GPA2/GPB5 influences the expression of these other receptor transcripts. Downregulating LGR1 results in a smaller blood meal size and disrupts the normal time-course of diuresis. As LGR1 is the most abundantly expressed G protein-coupled receptor transcript in R. prolixus MTs, our results suggest that GPA2/GPB5 signaling has a critical role in regulating the timing and success of water retention in the unfed state, and in the complex processes associated with feeding and diuresis in R. prolixus.

RhoprCAPA-2 acts as a gonadotropin regulating reproduction in adult female, Rhodnius prolixus.

CAPA peptides play diverse roles in insects, modulating muscle contraction, regulating fluid balance, and reproduction. In Rhodnius prolixus, a hematophagous insect and a vector for human Chagas disease, three CAPA peptides are encoded by the capability gene, including RhoprCAPA-1, RhoprCAPA-2, and RhoprCAPA-PK-1. RhoprCAPA-2 is an anti-diuretic hormone in R. prolixus. Here, we explore the involvement of RhoprCAPA-2 in reproduction in adult female R. prolixus. Double-label immunohistochemistry reveals co-localization of RhoprCAPA-2-like and the glycoprotein hormone (GPA2/GPB5) subunit GPB5-like immunoreactivity in neurosecretory cells in the mesothoracic ganglionic mass and in their neurohemal sites, suggesting these peptides can be co-released to regulate physiological processes. qPCR analysis reveals changes in transcript expression levels of the RhoprCAPA receptor (CAPAR) in the fat body and reproductive tissues after feeding in adult female R. prolixus. RNA interference-mediated knockdown of CAPAR transcript decreases egg production and reduces hatching rate and survival rate in female R. prolixus. Downregulation of CAPAR decreases vitellogenin RhoprVg1 transcript expression in the fat body and deceases its receptor RhoprVgR transcript level in the ovaries; accompanied by a reduction in vitellogenin content in the fat body and hemolymph. Incubation of fat body and ovaries in vitro with RhoprCAPA-2 increases RhoprVg1 transcript expression in the fat body, vitellogenin content in the fat body culture medium, and increases RhoprVgR transcript in the ovaries. These findings implicate the CAPA signaling pathway in reproduction, with RhoprCAPA-2 acting as a gonadotropin in adult female R. prolixus.

The role of ecdysteroid in the regulation of ovarian growth and oocyte maturation in Rhodnius prolixus, a vector of Chagas disease.

Rhodnius prolixus is a blood-gorging insect that is medically important since it transmits Chagas disease via feces and urine that contain the parasite Trypanosoma cruzi. In adult females, ecdysteroid hormone (20-hydroxyecdysone, 20E) is involved in the growth of the ovary and development of eggs post-blood meal (PBM). Halloween genes are essential for ecdysteroid synthesis since they code for cytochrome P450 enzymes in the ecdysteroidogenic pathway. The ecdysone receptor (EcR/USP) binds 20E, resulting in activation of ecdysone-responsive genes. We have identified and characterized the Halloween genes, and the non-Halloween gene, neverland, in the R. prolixus ovary using transcriptomic data. We used BLAST to compare transcriptome sequences with other arthropod sequences to identify similar transcripts. Our results indicate that the Halloween genes, neverland and ecdysone receptor transcripts are present in the ovaries of R. prolixus. We have quantified, by qPCR, Halloween gene transcript expression in the ovary following a blood meal. Most of the Halloween genes are upregulated during the first 3 days PBM. Knockdown of EcR, USP and shade transcripts, using RNA interference, results in a significant reduction in the number of eggs produced and a severe reduction in egg laying and hatching rate. Furthermore, knockdown of the EcR or shade transcripts altered the expression of the chorion gene transcripts Rp30 and Rp45 at day 3 and 6 PBM. These results indicate that ecdysteroids play critical roles in reproduction of female R. prolixus.

The hormonal and neural control of egg production in the historically important model insect, Rhodnius prolixus: A review, with new insights in this post-genomic era.

Rhodnius prolixus, the blood gorging kissing bug, is a model insect, extensively used by Sir Vincent Wigglesworth and others, upon which the foundations of insect physiology, endocrinology, and development are built. It is also medically important, being a principal vector of Trypanosoma cruzi, the causative agent of Chagas disease in humans. The blood meal stimulates and enables egg production, and since an adult mated female can take several blood meals, each female can produce hundreds of offspring. Understanding the reproductive biology of R. prolixus is therefore of some critical importance for controlling the transmission of Chagas disease. The R. prolixus genome is available and so the post-genomic era has arrived for this historic model insect. This review focuses on the female reproductive system and coordination over the production of eggs, emphasizing the classical (neuro)endocrinological studies that led to a model describing inputs from feeding and mating, and the neural control of egg-laying. We then review recent insights brought about by molecular analyses, including transcriptomics, that confirm, support, and considerably extends this model. We conclude this review with an updated model describing the events leading to full expression of egg production, and also provide a consideration of questions for future exploration and experimentation.

Impact of JH Signaling on Reproductive Physiology of the Classical Insect Model, Rhodnius prolixus.

In adult females of several insect species, juvenile hormones (JHs) act as gonadotrophic hormones, regulating egg production. JH binds to its nuclear receptor, Methoprene tolerant (Met), triggering its dimerization with the protein Taiman (Tai). The resulting active complex induces transcription of JH response genes, such as Krüppel homolog 1 (Kr-h1). In this study we report for the first time the participation of the isoform JH III skipped bisepoxide (JHSB3) and its signaling pathway in the reproductive fitness of the classical insect model Rhodnius prolixus. The topical application of synthetic JHSB3 increases transcript and protein expression of yolk protein precursors (YPPs), mainly by the fat body but also by the ovaries, the second source of YPPs. These results are also confirmed by ex vivo assays. In contrast, when the JH signaling cascade is impaired via RNA interference by downregulating RhoprMet and RhoprTai mRNA, egg production is inhibited. Although RhoprKr-h1 transcript expression is highly dependent on JHSB3 signaling, it is not involved in egg production but rather in successful hatching. This research contributes missing pieces of JH action in the insect model in which JH was first postulated almost 100 years ago.

Crosstalk between Nutrition, Insulin, Juvenile Hormone, and Ecdysteroid Signaling in the Classical Insect Model, Rhodnius prolixus.

The rigorous balance of endocrine signals that control insect reproductive physiology is crucial for the success of egg production. Rhodnius prolixus, a blood-feeding insect and main vector of Chagas disease, has been used over the last century as a model to unravel aspects of insect metabolism and physiology. Our recent work has shown that nutrition, insulin signaling, and two main types of insect lipophilic hormones, juvenile hormone (JH) and ecdysteroids, are essential for successful reproduction in R. prolixus; however, the interplay behind these endocrine signals has not been established. We used a combination of hormone treatments, gene expression analyses, hormone measurements, and ex vivo experiments using the corpus allatum or the ovary, to investigate how the interaction of these endocrine signals might define the hormone environment for egg production. The results show that after a blood meal, circulating JH levels increase, a process mainly driven through insulin and allatoregulatory neuropeptides. In turn, JH feeds back to provide some control over its own biosynthesis by regulating the expression of critical biosynthetic enzymes in the corpus allatum. Interestingly, insulin also stimulates the synthesis and release of ecdysteroids from the ovary. This study highlights the complex network of endocrine signals that, together, coordinate a successful reproductive cycle.

Identification and cloning of the kinin receptor in the Chagas disease vector, Rhodnius prolixus.

Within invertebrates, the kinin family of neuropeptides is responsible for the modulation of a host of physiological and behavioural processes. In Rhodnius prolixus, kinins are primarily responsible for eliciting myotropic effects on various feeding and diuresis-related tissues. Here, the R. prolixus kinin receptor (RhoprKR) has been identified, cloned and sequenced from the central nervous system (CNS) and hindgut of R. prolixus. Sequence analyses show high similarity and identity between RhoprKR and other cloned invertebrate kinin receptors. The expression profile of RhoprKR shows the RhoprKR transcript throughout the R. prolixus gut, with highest expression in the hindgut, suggesting a role of Rhopr-kinins in various aspects of feeding and digestion. RNA interference (RNAi)-mediated knockdown of the RhoprKR transcript resulted in a significant reduction of hindgut contractions in response to Rhopr-kinin 2 and an Aib-containing kinin analog. dsRhoprKR- injected insects also consumed a significantly larger meal, suggesting a role of Rhopr-kinins in satiety.

Jaburetox, a natural insecticide derived from Jack Bean Urease, activates voltage-gated sodium channels to modulate insect behavior.

Jaburetox (Jbtx) is an insecticidal peptide derived from Canavalia ensiformis urease, whose mechanism of action is not completely elucidated. We employed behavioral, electromyographical and electrophysiological protocols to identify the cellular and molecular targets involved in the Jbtx entomotoxicity in cockroaches and locusts. In Nauphoeta cinerea, Jbtx (32 µg/g) altered the locomotory behaviour inducing a significative decrease in the distance travelled followed by a significant increase in stopped time (52 ±â€¯85 cm and 2573 ±â€¯89 s, p < .05, n = 40). Jbtx (8 to 32 µg/g body weight, respectively) also increased the leg and antennae grooming activities (p < .05, n = 40, respectively). Jbtx (8 to 16 µg/g) induced a maximum neuromuscular blockade of 80.72% (n = 6, p < .05) and was cardiotoxic, decreasing the cockroach heart rate. The electrophysiological profiles of both muscle and nerve of L. migratoria showed that Jbtx (2.5 × 10-7 and 2.5 × 10-3 µg/ body weight) induced a significant increase in the amplitude of nerve action potentials (n = 5, p < .05). Voltage clamp analysis of Jbtx (200 nM) applied in Xenopus laevis oocytes heterologously expressed with Nav 1.1 channels showed a significant increase in the sodium currents. In conclusion, this work revealed that the entomotoxic activity of Jbtx involves complex behavioral alterations that begins with an initial activation of voltage-gated sodium channels.

The involvement of Rhopr-CRF/DH in feeding and reproduction in the blood-gorging insect Rhodnius prolixus.

Rhodnius prolixus is a blood-gorging insect and a vector for human Chagas disease. The insect transmits the disease following feeding, when it excretes urine and feces contaminated with the Trypanosoma cruzi parasite. A corticotropin-releasing factor-like peptide acts as a diuretic hormone in R. prolixus (Rhopr-CRF/DH); however, its distribution throughout the insect's central nervous system (CNS) and the expression of its receptor in feeding-related tissue as well as the female reproductive system suggests a multifaceted role for the hormone beyond that of diuresis. Here we investigate the involvement of Rhopr-CRF/DH in feeding and reproduction in R. prolixus. Immunohistochemistry of the CNS showed diminished CRF-like staining in neurosecretory cells (NSCs) of the mesothoracic ganglionic mass (MTGM) immediately following feeding, and partial restocking of those same cells two hours later, indicating Rhopr-CRF/DH stores in this regions are involved in feeding. The results of the temporal qPCR analysis were consistent with the immunohistochemical findings, showing an increase in Rhopr-CRF/DH transcript expression in the MTGM immediately after feeding, presumably capturing the restocking of Rhopr-CRF/DH in the lateral NSCs following release of the peptide during feeding. Elevating haemolymph Rhopr-CRF/DH titres by injection of Rhopr-CRF/DH prior to feeding resulted in the intake of a significantly smaller blood meal in 5th instars and adults without an apparent effect on the rate of short-term diuresis. When adult females were injected with Rhopr-CRF/DH, they also produced and laid significantly fewer eggs. Finally, in vitro oviduct contraction assays illustrate that Rhopr-CRF/DH inhibits the amplitude of contractions of the lateral oviducts, highlighting a potential mechanism via which the hormone diminishes reproductive capacity. To conclude, the study of the Rhopr-CRF/DH pathway, its components and mechanisms of action, has implications for vector control by highlighting targets to alter feeding, diuresis, and reproduction of this disease vector.

Jack bean urease modulates neurotransmitter release at insect neuromuscular junctions.

BACKGROUND: Plants have developed a vast range of mechanisms to compete with phytophagous insects, including entomotoxic proteins such as ureases. The legume Canavalia ensiformis produces several urease isoforms, of which the more abundant is called Jack Bean Urease (JBU). Previews work has demonstrated the potential insecticidal effects of JBU, by mechanisms so far not entirely elucidated. In this work, we investigated the mechanisms involved in the JBU-induced activity upon neurotransmitter release on insect neuromuscular junctions. METHODS: Electrophysiological recordings of nerve and muscle action potentials, and calcium imaging bioassays were employed. RESULTS AND CONCLUSION: JBU (0.28 mg/animal/day) in Locusta migratoria 2nd instar through feeding and injection did not induce lethality, although it did result in a reduction of 20% in the weight gain at the end of 168 h (n = 9, p ≤ 0.05). JBU (0.014 and 0.14 mg) injected direct into the locust hind leg induced a dose and time-dependent decrease in the amplitude of muscle action potentials, with a maximum decrease of 70% in the amplitude at the highest dose (n = 5, p ≤ 0.05). At the same doses JBU did not alter the amplitude of action potentials evoked from motor neurons. Using Drosophila 3rd instar larvae neuromuscular preparations, JBU (10-7 M) increased the occurrence of miniature Excitatory Junctional Potentials (mEJPs) in the presence of 1 mM CaCl2 (n = 5, p ≤ 0.05). In low calcium (0.4 mM) assays, JBU (10-7 M) was not able to modulate the occurrence of the events. In Ca2+-free conditions, with EGTA or CoCl2, JBU induced a significant decrease in the occurrence of mEPJs (n = 5, p ≤ 0.05). Injected into the 3rd abdominal ganglion of Nauphoeta cinerea cockroaches, JBU (1 µM) induced a significant increase in Ca2+ influx (n = 7, p ≤ 0.01), similar to that seen for high KCl (35 mM) condition. Taken together the results confirm a direct action of JBU upon insect neuromuscular junctions and possibly central synapses, probably by disrupting the calcium machinery in the pre-synaptic region of the neurons.

Octopamine and tyramine regulate the activity of reproductive visceral muscles in the adult female blood-feeding bug, Rhodnius prolixus.

The role of octopamine and tyramine in regulating spontaneous contractions of reproductive tissues was examined in the female Rhodnius prolixus Octopamine decreased the amplitude of spontaneous contractions of the oviducts and reduced RhoprFIRFa-induced contractions in a dose-dependent manner, whereas tyramine only reduced the RhoprFIRFa-induced contractions. Both octopamine and tyramine decreased the frequency of spontaneous bursal contractions and completely abolished the contractions at 5×10-7 mol l-1 and above. Phentolamine, an octopamine receptor antagonist, attenuated the inhibition induced by octopamine on the oviducts and the bursa. Octopamine also increased the levels of cAMP in the oviducts, and this effect was blocked by phentolamine. Dibutyryl cyclic AMP mimicked the effects of octopamine by reducing the frequency of bursal contractions, suggesting that the octopamine receptor may act by an Octß receptor. The tyramine receptor antagonist yohimbine failed to block the inhibition of contractions induced by tyramine on the bursa, suggesting that tyramine may be acting on the Octß receptor in the bursa.

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.

K⁺ absorption by locust gut and inhibition of ileal K⁺ and water transport by FGLamide allatostatins.

The scanning ion-selective electrode technique (SIET) was utilized for the first time in Locusta migratoria to characterize K(+) transport along the digestive tract and to determine the effect of two locust FGLamide allatostatins (FGLa/ASTs) on K(+) transport: a previously sequenced FGLa/AST from Schistocerca gregaria (Scg-AST-6; ARPYSFGL-NH2) and a newly sequenced FGLa/AST from L. migratoria (Locmi-FGLa/AST-2; LPVYNFGL-NH2). Regional differences in K(+) fluxes along the gut were evident, where K(+) efflux in vitro (or absorption into the hemolymph in vivo) was greatest at the anterior ileum, and lowest at the colon. Ileal K(+) efflux was inhibited by both Scg-AST-6 and Locmi-FGLa/AST-2, with maximal inhibition at 10(-10) and 10(-11) mol l(-1), respectively. Both FGLa/ASTs also inhibited cAMP-stimulated K(+) efflux from the ileum. Locmi-FGLa/AST-2 also inhibited efflux of water across the ileum. Locusts are terrestrial insects living in dry climates, risking desiccation and making water conservation a necessity. The results suggest that FGLa/ASTs may be acting as diuretics by increasing K(+) excretion and therefore increasing water excretion. Thus it is likely that FGLa/ASTs are involved in the control of hemolymph water and ion levels during feeding and digestion, to help the locust deal with the excess K(+) load (and subsequently fluid) when the meal is processed.

The neuroendocrine and endocrine systems in insect - Historical perspective and overview.

A complex cascade of events leads to the initiation and maintenance of a behavioral act in response to both internally and externally derived stimuli. These events are part of a transition of the animal into a new behavioral state, coordinated by chemicals that bias tissues and organs towards a new functional state of the animal. This form of integration is defined by the neuroendocrine (or neurosecretory) system and the endocrine system that release neurohormones or hormones, respectively. Here we describe the classical neuroendocrine and endocrine systems in insects to provide an historic perspective and overview of how neurohormones and hormones support plasticity in behavioral expression. Additionally, we describe peripheral tissues such as the midgut, epitracheal glands, and ovaries, which, whilst not necessarily being endocrine glands in the pure sense of the term, do produce and release hormones, thereby providing even more flexibility for inter-organ communication and regulation.

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.

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.

A critical role for ecdysone response genes in regulating egg production in adult female Rhodnius prolixus.

Ecdysteroids control ovary growth and egg production through a complex gene hierarchy. In the female Rhodnius prolixus, a blood-gorging triatomine and the vector of Chagas disease, we have identified the ecdysone response genes in the ovary using transcriptomic data. We then quantified the expression of the ecdysone response gene transcripts (E75, E74, BR-C, HR3, HR4, and FTZ-F1) in several tissues, including the ovary, following a blood meal. These results confirm the presence of these transcripts in several tissues in R. prolixus and show that the ecdysone response genes in the ovary are mostly upregulated during the first three days post blood meal (PBM). Knockdown of E75, E74, or FTZ-F1 transcripts using RNA interference (RNAi) was used to understand the role of the ecdysone response genes in vitellogenesis and egg production. Knockdown significantly decreases the expression of the transcripts for the ecdysone receptor and Halloween genes in the fat body and the ovaries and reduces the titer of ecdysteroid in the hemolymph. Knockdown of each of these transcription factors typically alters the expression of the other transcription factors. Knockdown also significantly decreases the expression of vitellogenin transcripts, Vg1 and Vg2, in the fat body and ovaries and reduces the number of eggs produced and laid. Some of the laid eggs have an irregular shape and smaller volume, and their hatching rate is decreased. Knockdown also influences the expression of the chorion gene transcripts Rp30 and Rp45. The overall effect of knockdown is a decrease in number of eggs produced and a severe reduction in number of eggs laid and their hatching rate. Clearly, ecdysteroids and ecdysone response genes play a significant role in reproduction in R. prolixus.

The Male Reproductive System of the Kissing Bug, Rhodnius prolixus Stål, 1859 (Hemiptera: Reduviidae: Triatominae): Arrangements of the Muscles and the Myoactivity of the Selected Neuropeptides.

The gross anatomy of the male reproductive structures and their associated musculature are described in the blood-gorging vector of Chagas disease, Rhodnius prolixus. The male reproductive system is composed of muscular tissues each performing contractions that aid in the movement of sperm out of the testis into the vas deferens, seminal vesicle and then into the ejaculatory duct, along with proteins and lipids from the transparent and opaque accessory glands. Phalloidin staining shows the various patterns of muscle fiber layers, from thin circular to more complex crisscross patterns, implying subtle differences in the form of the contractions and movement of each of the structures, allowing for waves of contractions or twisting patterns. The transcripts for the receptors for proctolin, myosuppressin (RhoprMS) and for the extended FMRFamides are expressed in the various regions of the reproductive system, and the nerve processes extending over the reproductive structures are positive for FMRFamide-like immunoreactivity, as are neurosecretory cells lying on the nerves. Proctolin and AKDNFIRFamide are strong stimulators for the frequency of the contractions, and RhoprMS can inhibit the proctolin-induced contractions. Taken together, this work implicates these two families of peptides in coordinating the male reproductive structures for the successful transfer of sperm and the associated accessory gland fluid to the female during copulation.

Tyraminergic control of vitellogenin production and release in the blood-feeding insect, Rhodnius prolixus.

In insects, the biogenic amine tyramine (TA) has been shown to control several physiological processes. Recently, the involvement of the type 1 tyramine receptor (TAR1) in reproductive processes has been demonstrated in different insects. Here, we investigate the putative role of Rhodnius prolixus TAR1 (RpTAR1) in reproduction in female R. prolixus. RpTAR1 transcript was highly expressed in tissues associated with egg development. Moreover, after a blood meal, which is the stimulus for full egg development, RpTAR1 transcript was upregulated in the ovaries and in the fat body. After RNAi-mediated RpTAR1 knockdown, an ovarian phenotype characterized by the absence or reduction of egg production was observed. Furthermore, protein and Vg accumulation in the fat body was observed, suggesting an impairment in protein release from the fat body into the hemolymph. However, even though fewer eggs were produced and laid, there was no difference in hatching ratio of those laid, in comparison to the controls, indicating that the overall low protein uptake by the ovaries did not influence the viability of individual eggs produced. Interestingly, the eggs from dsTAR1-treated insects appeared more red, indicating a higher content of RHBP compared to the control. A higher colocalization between Vg and Rab11, a marker for the recycling endosome pathway, was observed after dsTAR1 injection, suggesting that a more active lysosome degradation pathway in response to the Vg accumulation may occur. In addition to the Vg accumulation in the fat body, dsTAR1 treatment altered JH pathway. However, it remains to be elucidated whether this event is either directly related to the RpTAR1 downregulation or for a consequence to the Vg accumulation. Lastly, the RpTAR1 action on Vg synthesis and release in the fat body was monitored in the presence or absence of yohimbine, the antagonist of TAR1, in an ex-vivo experiment. Yohimbine antagonises the TAR1 stimulated release of Vg. These results provide critical information concerning the role of TAR1 in Vg synthesis and release in R. prolixus. Furthermore, this work opens the way for further investigation into innovative methods for controlling R. prolixus.