Trypanosoma cruzi reprograms mitochondrial metabolism within the anterior midgut of its vector Rhodnius prolixus during the early stages of infection.

BACKGROUND: Trypanosoma cruzi is transmitted to humans by hematophagous bugs belonging to the Triatominae subfamily. Its intra-vectorial cycle is complex and occurs exclusively in the insect's midgut. Dissecting the elements involved in the cross-talk between the parasite and its vector within the digestive tract should provide novel targets for interrupting the parasitic life cycle and affecting vectorial competence. These interactions are shaped by the strategies that parasites use to infect and exploit their hosts, and the host's responses that are designed to detect and eliminate parasites. The objective of the current study is to characterize the impact of T. cruzi establishment within its vector on the dynamics of its midgut. METHODS: In this study, we evaluated the impact of T. cruzi infection on protein expression within the anterior midgut of the model insect Rhodnius prolixus at 6 and 24 h post-infection (hpi) using high-throughput quantitative proteomics. RESULTS: Shortly after its ingestion, the parasite modulates the proteome of the digestive epithelium by upregulating 218 proteins and negatively affecting the expression of 11 proteins involved in a wide array of cellular functions, many of which are pivotal due to their instrumental roles in cellular metabolism and homeostasis. This swift response underscores the intricate manipulation of the vector's cellular machinery by the parasite. Moreover, a more in-depth analysis of proteins immediately induced by the parasite reveals a pronounced predominance of mitochondrial proteins, thereby altering the sub-proteomic landscape of this organelle. This includes various complexes of the respiratory chain involved in ATP generation. In addition to mitochondrial metabolic dysregulation, a significant number of detoxifying proteins, such as antioxidant enzymes and P450 cytochromes, were immediately induced by the parasite, highlighting a stress response. CONCLUSIONS: This study is the first to illustrate the response of the digestive epithelium upon contact with T. cruzi, as well as the alteration of mitochondrial sub-proteome by the parasite. This manipulation of the vector's physiology is attributable to the cascade activation of a signaling pathway by the parasite. Understanding the elements of this response, as well as its triggers, could be the foundation for innovative strategies to control the transmission of American trypanosomiasis, such as the development of targeted interventions aimed at disrupting parasite proliferation and transmission within the triatomine vector.

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.

Export of heme by the feline leukemia virus C receptor regulates mitochondrial biogenesis and redox balance in the hematophagous insect Rhodnius prolixus.

Heme is a prosthetic group of proteins involved in vital physiological processes. It participates, for example, in redox reactions crucial for cell metabolism due to the variable oxidation state of its central iron atom. However, excessive heme can be cytotoxic due to its prooxidant properties. Therefore, the control of intracellular heme levels ensures the survival of organisms, especially those that deal with high concentrations of heme during their lives, such as hematophagous insects. The export of heme initially attributed to the feline leukemia virus C receptor (FLVCR) has recently been called into question, following the discovery of choline uptake by the same receptor in mammals. Here, we found that RpFLVCR is a heme exporter in the midgut of the hematophagous insect Rhodnius prolixus, a vector for Chagas disease. Silencing RpFLVCR decreased hemolymphatic heme levels and increased the levels of intracellular dicysteinyl-biliverdin, indicating heme retention inside midgut cells. FLVCR silencing led to increased expression of heme oxygenase (HO), ferritin, and mitoferrin mRNAs while downregulating the iron importers Malvolio 1 and 2. In contrast, HO gene silencing increased FLVCR and Malvolio expression and downregulated ferritin, revealing crosstalk between heme degradation/export and iron transport/storage pathways. Furthermore, RpFLVCR silencing strongly increased oxidant production and lipid peroxidation, reduced cytochrome c oxidase activity, and activated mitochondrial biogenesis, effects not observed in RpHO-silenced insects. These data support FLVCR function as a heme exporter, playing a pivotal role in heme/iron metabolism and maintenance of redox balance, especially in an organism adapted to face extremely high concentrations of heme.

Local age-dependent neuromodulation in Rhodnius prolixus antennae.

Kissing bugs do not respond to host cues when recently molted and only exhibit robust host-seeking several days after ecdysis. Behavioral plasticity has peripheral correlates in antennal gene expression changes through the week after ecdysis. The mechanisms regulating these peripheral changes are still unknown, but neuropeptide, G-protein coupled receptor, nuclear receptor, and takeout genes likely modulate peripheral sensory physiology. We evaluated their expression in antennal transcriptomes along the first week postecdysis of Rhodnius prolixus 5th instar larvae. Besides, we performed clustering and co-expression analyses to reveal relationships between neuromodulatory (NM) and sensory genes. Significant changes in transcript abundance were detected for 50 NM genes. We identified 73 sensory-related and NM genes that were assigned to nine clusters. According to their expression patterns, clusters were classified into four groups: two including genes up or downregulated immediately after ecdysis; and two with genes with expression altered at day 2. Several NM genes together with sensory genes belong to the first group, suggesting functional interactions. Co-expression network analysis revealed a set of genes that seem to connect with sensory system maturation. Significant expression changes in NM components were described in the antennae of R. prolixus after ecdysis, suggesting that a local NM system acts on antennal physiology. These changes may modify the sensitivity of kissing bugs to host cues during this maturation interval.

Unveiling the Peptidase Network Orchestrating Hemoglobin Catabolism in Rhodnius prolixus.

Chagas disease is transmitted to humans by obligatory hematophagous insects of Triatominae subfamily, which feeds on various hosts to acquire their nutritional sustenance derived from blood proteins. Hemoglobin (Hb) digestion is a pivotal metabolic feature of triatomines, representing a key juncture in their competence toward Trypanosoma cruzi; however, it remains poorly understood. To explore the Hb digestion pathway in Rhodnius prolixus, a major Chagas disease vector, we employed an array of approaches for activity profiling of various midgut-associated peptidases using specific substrates and inhibitors. Dissecting the individual contribution of each peptidase family in Hb digestion has unveiled a predominant role played by aspartic proteases and cathepsin B-like peptidases. Determination of peptidase-specific cleavage sites of these key hemoglobinases, in conjunction with mass spectrometry-based identification of in vivo Hb-derived fragments, has revealed the intricate network of peptidases involved in the Hb digestion pathway. This network is initiated by aspartic proteases and subsequently sustained by cysteine proteases belonging to the C1 family. The process is continued simultaneously by amino and carboxypeptidases. The comprehensive profiling of midgut-associated aspartic proteases by quantitative proteomics has enabled the accurate revision of gene annotations within the A1 family of the R. prolixus genome. Significantly, this study also serves to illuminate a potentially important role of the anterior midgut in blood digestion. The expanded repertoire of midgut-associated proteases presented in this study holds promise for the identification of novel targets aimed at controlling the transmission of Chagas disease.

Halloween genes are expressed with a circadian rhythm during development in prothoracic glands of the insect RHODNIUS PROLIXUS.

We analyse the developmental and circadian profiles of expression of the genes responsible for ecdysteroidogenesis (Halloween genes) in the PGs of Rhodnius prolixus throughout larval-adult development. Extensive use of in vitro techniques enabled multiple different parameters to be measured in individual PGs. Expression of disembodied and spook closely paralleled the ecdysteroid synthesis of the same PGs, and the ecdysteroid titre in vivo, but with functionally significant exceptions. Various tissues other than PGs expressed one, both or neither genes. Both gonads express both genes in pharate adults (larvae close to ecdysis). Both genes were expressed at low, but significant, levels in UF Rhodnius, raising questions concerning how developmental arrest is maintained in UF animals. IHC confirmed the subcellular localisation of the coded proteins. Gene knockdown suppressed transcription of both genes and ecdysteroid synthesis, with spook apparently regulating the downstream gene disembodied. Transcription of both genes occurred with a daily rhythm (with peaks at night) that was confirmed to be under circadian control using aperiodic conditions. The complex behaviour of the rhythm in LL implied two anatomically distinct oscillators regulate this transcription rhythm. First, the circadian clock in the PGs and second, the circadian rhythm of of Rhodnius PTTH which is released rhythmically from the brain under control of the circadian clock therein, both of which were described previously. We conclude ecdysteroidogenesis in Rhodnius PGs employs a similar pathway as other insects, but its control is complex, involving mechanisms both within and outside the PGs.

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.

Allatotropic peptides modulate muscle contraction of the female reproductive system in Rhodnius prolixus (STÄL).

Allatotropin (AT) acts as a myoregulator at the level of the dorsal vessel (DV) and midgut (MG) in triatominae insects. Previous analyses of the expression of the AT receptor in Rhodnius prolixus showed that AT is expressed in the DV and MG, but also in the reproductive system in females. To further study the activity of AT on female reproductive organs we analyzed the response by adult females in different physiological conditions, including unfed (virgin and mated), and fed mated females (gravid), to doses ranging between 10-14 and 10-6M. Myoregulatory activity was evaluated in vivo, by recording independently the frequency of contractions of each organ after treatment. The results show that the effect of AT varies depending on the organs and on the physiological state of the female. Whilst unfed virgin females did not show response to the peptide for neither of the applied doses, the ovaries showed a differential response, presenting the highest frequency of contractions in gravid individuals. An increase in the frequency of contractions of the oviducts was only observed in mated females. Uterus and spermathecae responded in both gravid and mated females, with maximum activity in the latter. In the bursa, responses were only detected in gravid females. The differential response of the organs seems to be associated to particular moments along the reproductive cycle, such as with the spermathecae that reacted to AT in both unfed mated and gravid females, when the movement of spermatozoids is physiologically crucial. Testes and accessory glands of the male, expressed the mRNA of AT precursor, suggesting that the male would modulate the contractile behavior of the female reproductive system after copula. The ovaries also expressed AT mRNA suggesting the existence of a paracrine/autocrine system modulating muscle contraction.

A pro-BMP function exerted by Rhodnius prolixus short gastrulation reveals great diversity in the role of BMP modulators during embryonic patterning.

Dorsal-ventral (DV) patterning is regulated by the bone morphogenetic pathway (BMP) in Bilateria. In insect DV patterning, the Toll pathway also plays a role, in addition to BMPs. Variations in the relative importance of each pathway for DV patterning have been reported using single species of coleopteran, hymenopteran, hemipteran and orthopteran insects. To investigate if the molecular control of DV patterning is conserved inside an insect order, the emergent model hemiptera species Rhodnius prolixus was studied. We found that R. prolixus BMP pathway controls the entire DV axis, with a broader effect respective to Toll, as shown for the hemiptera Oncopeltus fasciatus. Different from O. fasciatus, the unique R. prolixus short gastrulation (sog) and the twisted gastrulation (tsg) orthologues do not antagonize, but rather favour embryonic BMP signalling. Our results reinforce the hypothesis that hemiptera rely preferentially on BMPs for DV patterning but that, surprisingly, in R. prolixus Sog and Tsg proteins exert only a positive role to establish a dorsal-to-ventral BMP gradient. Since sog has been reported to be lost from orthopteran and hymenopteran genomes, our results indicate that Sog's role to modify BMP activity varies greatly in different insect species.

Heme protonation affects iron-NO binding in the NO transport protein nitrophorin.

The nitrophorins (NPs) comprise an unusual group of heme proteins with stable ferric heme iron nitric oxide (Fe-NO) complexes. They are found in the salivary glands of the blood-sucking kissing bug Rhodnius prolixus, which uses the NPs to transport the highly reactive signaling molecule NO. Nuclear resonance vibrational spectroscopy (NRVS) of both isoform NP2 and a mutant NP2(Leu132Val) show, after addition of NO, a strong structured vibrational band at around 600 cm-1, which is due to modes with significant Fe-NO bending and stretching contribution. Based on a hybrid calculation method, which uses density functional theory and molecular mechanics, it is demonstrated that protonation of the heme carboxyl groups does influence both the vibrational properties of the Fe-NO entity and its electronic ground state. Moreover, heme protonation causes a significant increase of the gap between the highest occupied and lowest unoccupied molecular orbital by almost one order of magnitude leading to a stabilization of the Fe-NO bond.

ATP synthase affects lipid metabolism in the kissing bug Rhodnius prolixus beyond its role in energy metabolism.

ATP synthase plays an essential role in mitochondrial metabolism, being responsible for the production of ATP in oxidative phosphorylation. However, recent results have shown that it may also be present in the cell membrane, involved in lipophorin binding to its receptors. Here, we used a functional genetics approach to investigate the roles of ATP synthase in lipid metabolism in the kissing bug Rhodnius prolixus. The genome of R. prolixus encodes five nucleotide-binding domain genes of the ATP synthase α and ß family, including the α and ß subunits of ATP synthase (RpATPSynα and RpATPSynß), and the catalytic and non-catalytic subunits of the vacuolar ATPase (RpVha68 and RpVha55). These genes were expressed in all analyzed organsn highest in the ovaries, fat body and flight muscle. Feeding did not regulate the expression of ATP synthases in the posterior midgut or fat body. Furthermore, ATP synthase is present in the fat body's mitochondrial and membrane fractions. RpATPSynß knockdown by RNAi impaired ovarian development and reduced egg-laying by approximately 85%. Furthermore, the lack of RpATPSynß increased the amount of triacylglycerol in the fat body due to increased de novo fatty acid synthesis and reduced transfer of lipids to lipophorin. RpATPSynα knockdown had similar effects, with altered ovarian development, reduced oviposition, and triacylglycerol accumulation in the fat body. However, ATP synthases knockdown had only a slight effect on the amount of ATP in the fat body. These results support the hypothesis that ATP synthase has a direct role in lipid metabolism and lipophorin physiology, which are not directly due to changes in energy metabolism.

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.

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 prothoracicotropic hormone (PTTH) of Rhodnius prolixus (Hemiptera) is noggin-like: Molecular characterisation, functional analysis and evolutionary implications.

Prothoracicotropic hormone (PTTH) is a central regulator of insect development that regulates the production of the steroid moulting hormones (ecdysteroids) from the prothoracic glands (PGs). Rhodnius PTTH was the first brain neurohormone discovered in any animal almost 100 years ago but has eluded identification and no homologue of Bombyx mori PTTH occurs in its genome. Here, we report Rhodnius PTTH is the first noggin-like PTTH found. It differs in important respects from known PTTHs and is the first PTTH from the Hemimetabola (Exopterygota) to be fully analysed. Recorded PTTHs are widespread in Holometabola but close to absent in hemimetabolous orders. We concluded Rhodnius PTTH likely differed substantially from the known ones. We identified one Rhodnius gene that coded a noggin-like protein (as defined by Molina et al., 2009) that had extensive similarities with known PTTHs but also had two additional cysteines. Sequence and structural analysis showed known PTTHs are closely related to noggin-like proteins, as both possess a growth factor cystine knot preceded by a potential cleavage site. The gene is significantly expressed only in the brain, in a few cells of the dorsal protocerebrum. We vector-expressed the sequence from the potential cleavage site to the C-terminus. This protein was strongly steroidogenic on PGs in vitro. An antiserum to the protein removed the steroidogenic protein released by the brain. RNAi performed on brains in vitro showed profound suppression of transcription of the gene and of production and release of PTTH and thus of ecdysteroid production by PGs. In vivo, the gene is expressed throughout development, in close synchrony with PTTH release, ecdysteroid production by PGs and the ecdysteroid titre. The Rhodnius PTTH monomer is 17kDa and immunoreactive to anti-PTTH of Bombyx mori (a holometabolan). Bombyx PTTH also mildly stimulated Rhodnius PGs. The two additional cysteines form a disulfide at the tip of finger 2, causing a loop of residues to protrude from the finger. A PTTH variant without this loop failed to stimulate PGs, showing the loop is essential for PTTH activity. It is considered that PTTHs of Holometabola evolved from a noggin-like protein in the ancestor of Holometabola and Hemiptera, c.400ma, explaining the absence of holometabolous-type PTTHs from hemimetabolous orders and the differences of Rhodnius PTTH from them. Noggin-like proteins studied from Hemiptera to Arachnida were homologous with Rhodnius PTTH and may be common as PTTHs or other hormones in lower insects.

Topical delivery of dsRNA in two hemipteran species: Evaluation of RNAi specificity and non-target effects.

Double-stranded (ds) RNA-based technologies could provide novel and potential tool for pest management with efficiency and specificity of action. However, before applying this technique in the field, it is necessary to identify effective delivery methods and evaluate the non-target effects that may occur. In this article, we evaluated the effectiveness of dsRNA by topical delivery on a species of great agricultural interest, Halyomorpha halys. The specificity of action of the dsRNA was also investigated in Rhodnius prolixus, an insect phylogenetically close to H. halys. Of the three investigated genes (putative ATPase N2B, ATPase, serine/threonine-protein phosphatase PP1-ß catalytic subunit, PP1, and IAP repeat-containing protein 7-B-like, IAP), IAP and ATPase were able to induce higher mortality in H. halys nymphs compared to the control, with specific concentrations for each gene targeted. However, when the same RNAs were topically delivered to both R. prolixus 2nd and 3rd instar nymphs, no gene silencing and mortality were observed. For this reason, to assess dsRNA application-mediated non-target effects, we injected both H. halys and R. prolixus specific dsRNA in R. prolixus 5th instar nymphs. When the dsRNA targeting H. halys IAP was microinjected into R. prolixus 5th instar nymphs, no mortality was observed, suggesting a strong RNAi specificity. Together, these data suggest that the topical delivery could be suitable for the dsRNA to control H. halys population. Furthermore, its specificity of action would allow treatments towards single harmful species with limited non-target effects.

Empty-spiracles is maternally expressed and essential for neurodevelopment and early embryo determination in Rhodnius prolixus.

Since empty-spiracles (ems) was identified and characterized in Drosophila melanogaster as a head-gap gene, several studies have been carried out in other insect orders to confirm its evolutionary conserved function. Using the blood-sucking bug Rhodnius prolixus as biological model, we found an ems transcript with three highly conserved regions: Box-A, Box-B, and the homeodomain. R. prolixus embryos silenced by parental RNAi for two of these ems conserved regions showed both maternal and zygotic defects. Rp-emsB fragment results in early lethal embryogenesis, with eggs without any embryonic structure inside. Rp-emsB expression pattern is only maternally expressed and localized in the ovary tropharium, follicular cells, and in the unfertilized female pronucleus. Rp-emsA fragment is zygotically expressed during early blastoderm formation until late developmental stages in two main patterns: anterior in the antennal segment, and in a segmentary in the neuroblast and tracheal pits. R. prolixus knockdown embryos for Rp-emsA showed an incomplete larval hatching, reduced heads, and severe neuromotor defects. Furthermore, in situ hybridization revealed a spatial and temporal expression pattern that highly correlates with Rp-ems observed function. Here,Rp-ems function in R. prolixus development was validated, showing that empty-spiracles does not act as a true head-gap gene, but it is necessary for proper head development and crucial for early embryo determination and neurodevelopment.

Dynamics of maternal gene expression in Rhodnius prolixus.

The study of developmental processes in Rhodnius prolixus has recently advanced with the sequencing of the genome. In this work, we analyze the maternal gene expression driving oogenesis and early embryogenesis in R. prolixus. We examined the transcriptional profile of mRNAs to establish the genes expressed across the ovary, unfertilized eggs and different embryonic stages of R. prolixus until the formation of the germ band anlage (0, 12, 24, and 48 h post egg laying). We identified 81 putative maternal and ovary-related genes and validated their expression by qRT-PCR. We validate the function of the ortholog gene Bicaudal-D (Rp-BicD) by in situ hybridization and parental RNAi. Consistent with a role in oogenesis and early development of R. prolixus, we show that lack of Rp-BicD does not significantly affect oogenesis but impairs the formation of the blastoderm. Based on our findings, we propose three times of action for maternal genes during oogenesis and embryogenesis in 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.

Identification of a tachykinin receptor and its implication in carbohydrate and lipid homeostasis in Rhodnius prolixus, a chagas disease vector.

Neuropeptides and their receptors are fundamentally important in regulating many physiological and behavioural processes in insects. In this work, we have identified, cloned, and sequenced the tachykinin receptor (Rhopr-TKR) from Rhodnius prolixus, a vector of Chagas disease. The receptor is a G protein-coupled receptor belonging to the Rhodopsin Family A. The total length of the open reading frame of the Rhopr-TKR transcript is 1110 bp, which translates into a receptor of 338 amino acids. Fluorescent in-situ RNA-hybridization (FISH) for the Rhopr-TKR transcript shows a signal in a group of six bilaterally paired neurons in the protocerebrum of the brain, localized in a similar region as the insulin producing cells. To examine the role of tachykinin signaling in lipid and carbohydrate homeostasis we used RNA interference. Downregulation of the Rhopr-TKR transcript led to a decrease in the size of blood meal consumed and a significant increase in circulating carbohydrate and lipid levels. Further investigation revealed a close relationship between tachykinin and insulin signaling since the downregulation of the Rhopr-TKR transcript negatively affected the transcript expression for insulin-like peptide 1 (Rhopr-ILP1), insulin-like growth factor (Rhopr-IGF) and insulin receptor 1 (Rhopr-InR1) in both the central nervous system and fat body. Taken together, these findings suggest that tachykinin signaling regulates lipid and carbohydrate homeostasis via the insulin signaling pathway.