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.

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.

Population genomics and geographic dispersal in Chagas disease vectors: Landscape drivers and evidence of possible adaptation to the domestic setting.

Accurate prediction of vectors dispersal, as well as identification of adaptations that allow blood-feeding vectors to thrive in built environments, are a basis for effective disease control. Here we adopted a landscape genomics approach to assay gene flow, possible local adaptation, and drivers of population structure in Rhodnius ecuadoriensis, an important vector of Chagas disease. We used a reduced-representation sequencing technique (2b-RADseq) to obtain 2,552 SNP markers across 272 R. ecuadoriensis samples from 25 collection sites in southern Ecuador. Evidence of high and directional gene flow between seven wild and domestic population pairs across our study site indicates insecticide-based control will be hindered by repeated re-infestation of houses from the forest. Preliminary genome scans across multiple population pairs revealed shared outlier loci potentially consistent with local adaptation to the domestic setting, which we mapped to genes involved with embryogenesis and saliva production. Landscape genomic models showed elevation is a key barrier to R. ecuadoriensis dispersal. Together our results shed early light on the genomic adaptation in triatomine vectors and facilitate vector control by predicting that spatially-targeted, proactive interventions would be more efficacious than current, reactive approaches.

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.

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.

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.

Transovarial transmission of a core virome in the Chagas disease vector Rhodnius prolixus.

Triatomine assassin bugs comprise hematophagous insect vectors of Trypanosoma cruzi, the causative agent of Chagas disease. Although the microbiome of these species has been investigated to some extent, only one virus infecting Triatoma infestans has been identified to date. Here, we describe for the first time seven (+) single-strand RNA viruses (RpV1-7) infecting Rhodnius prolixus, a primary vector of Chagas disease in Central and South America. We show that the RpVs belong to the Iflaviridae, Permutotetraviridae and Solemoviridae and are vertically transmitted from the mothers to the progeny via transovarial transmission. Consistent with this, all the RpVs, except RpV2 that is related to the entomopathogenic Slow bee paralysis virus, established persistent infections in our R. prolixus colony. Furthermore, we show that R. prolixus ovaries express 22-nucleotide viral siRNAs (vsiRNAs), but not viral piRNAs, that originate from the processing of dsRNA intermediates during viral replication of the RpVs. Interestingly, the permutotetraviruses and sobemoviruses display shared pools of vsiRNAs that might provide the basis for a cross-immunity system. The vsiRNAs are maternally deposited in the eggs, where they likely contribute to reduce the viral load and protect the developing embryos. Our results unveil for the first time a complex core virome in R. prolixus and begin to shed light on the RNAi-based antiviral defenses in triatomines.

Using axenic and gnotobiotic insects to examine the role of different microbes on the development and reproduction of the kissing bug Rhodnius prolixus (Hemiptera: Reduviidae).

Kissing bugs (Hempitera: Reduviidae) are obligately and exclusively blood feeding insects. Vertebrate blood is thought to provide insufficient B vitamins to insects, which rely on symbiotic relationships with bacteria that provision these nutrients. Kissing bugs harbour environmentally acquired bacteria in their gut lumen, without which they are unable to develop to adulthood. Rhodococcus rhodnii was initially identified as the sole symbiont of Rhodnius prolixus, but modern studies of the kissing bug microbiome suggest that R. rhodnii is not always present or abundant in wild-caught individuals. We asked whether R. rhodnii or other bacteria alone could function as symbionts of R. prolixus. We produced insects with no bacteria (axenic) or with known microbiomes (gnotobiotic). Gnotobiotic insects harbouring R. rhodnii alone developed faster, had higher survival, and laid more eggs than those harbouring other bacterial monocultures, including other described symbionts of kissing bugs. R. rhodnii grew to high titre in the guts of R. prolixus while other tested species were found at much lower abundance. Rhodococcus species tested had nearly identical B vitamin biosynthesis genes, and dietary supplementation of B vitamins had a relatively minor effect on development and survival of gnotobiotic R. prolixus. Our results indicate that R. prolixus have a higher fitness when harbouring R. rhodnii than other bacteria tested, that this may be due to R. rhodnii existing at higher titres and providing more B vitamins to the host, and that symbiont B vitamin synthesis is probably a necessary but not sufficient function of gut bacteria in kissing bugs.

Investigation of Rhodnius prolixus head anatomy using microtomography.

In the last years, microtomography has proved to be a powerful technique on insects' studies, allowing a detailed view of the structures' internal with a high resolution. One of the most important advantages about the use of microtomography in these studies is the fact that the dissection is not necessary, which decreases considerably the number of samples used on the insects' research. Some insects are used constantly in studies about morphology, metamorphosis, and reproduction, because they work as a model for others, and Rhodnius prolixus is one of the most studied in this group. This insect is also one of the main insect vectors of Chagas disease that kills around 12,000 people every year in Latin America. Some studies using laboratory microtomography conventional scanners combining with the correct staining methods have proved that it could be a powerful tool in biological research, allowing the visualisation of low-density tissues. The main goal of the present work was to use staining protocols to study Rhodnius prolixus with laboratory microtomography conventional scanners. The experiments were carried out at the imaging lab in the Theoretical Biology Department, University of Vienna, using an Xradia MicroXCT and at the University of Oslo, using a Skyscan 2211.

Shelter selection in the kissing bugs Triatoma infestans and Rhodnius prolixus: Intra- and inter-specific interactions and quality evaluation.

Triatomines display most of their activities during the night. Before sunrise, they search, select and occupy adequate shelters to stay during the photophase, avoiding exposure to diurnal predators. In this work, we first explored the interactions between individuals of the same or different species in the shelter selection process of Triatoma infestans (Klug 1834) and Rhodnius prolixus (Stål 1859). When two groups of insects (either of different nutritional status, nymphal instar or species) were released together over an experimental arena containing two identical shelters, all nymphs were distributed randomly, suggesting the absence of intra- and inters-pecific interactions. Secondly, we analysed their preferences for particular features of shelters by releasing one group of insects (either T. infestans or R. prolixus) over an arena containing two different refuges. Nymphs exhibited preferences for darker shelters with a vertical orientation of its substrate and elevated from the ground, highlighting the importance of such features in a shelter selection context. We conclude that these species disregard the presence of other individuals but evaluate certain characteristics of the shelters to choose them. This information may contribute to understanding the colonization/recolonization dynamic processes of these Chagas disease vectors.

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.

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.

Population size regulation is density-dependent in Rhodnius prolixus (Hemiptera: Reduviidae) through an irritability mechanism.

BACKGROUND: Physical factors can determine the level of triatomine abundance, but do not regulate their population densities, and neither do natural enemies. OBJECTIVES: To identify the processes associated with density-dependent triatomine population regulation. METHODS: We set-up a laboratory experiment with four interconnected boxes; the central box harbored Rhodnius prolixus bugs and one hamster. Stage 5 and adult densities of 10, 20, 30, 40, and 60 bugs per hamster, were replicated four times (except the density of 60 bugs). Hamster's irritability and several triatomine responses were measured: feeding, development time and longevity, mortality, fecundity, dispersal, and the net reproductive value (R o ). FINDINGS: Density had a statistically significant effect on irritability, but not on the percent of bugs feeding. Density was significant on blood meal size ingested in bugs that did not move between boxes, but not significant when the bugs moved. Density and irritability affected the proportion of stage 5 nymphs molting, and the proportion of adult bugs dying per day and over a three-week period. There was a highly significant effect of density and irritability on R o . MAIN CONCLUSIONS: We showed that a density-dependent mechanism, acting through the irritability of the host, seems the most plausible process regulating populations in triatomines.

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.

Knockdown of E1- and E2-ubiquitin enzymes triggers defective chorion biogenesis and modulation of autophagy-related genes in the follicle cells of the vector Rhodnius prolixus.

In insects, the last stage of oogenesis is the process where the chorion layers (eggshell) are synthesized and deposited on the surface of the oocytes by the follicle cells. Protein homeostasis is determined by the fine-tuning of translation and degradation pathways, and the ubiquitin-proteasome system is one of the major degradative routes in eukaryotic cells. The conjugation of ubiquitin to targeted substrates is mediated by the ordered action of E1-activating, E2-conjugating, and E3-ligase enzymes, which covalently link ubiquitin to degradation-targeted proteins delivering them to the proteolytic complex proteasome. Here, we found that the mRNAs encoding polyubiquitin (pUbq), E1, and E2 enzymes are highly expressed in the ovaries of the insect vector of Chagas Disease Rhodnius prolixus. RNAi silencing of pUbq was lethal whereas the silencing of E1 and E2 enzymes resulted in drastic decreases in oviposition and embryo viability. Eggs produced by the E1- and E2-silenced insects presented particular phenotypes of altered chorion ultrastructure observed by high-resolution scanning electron microscopy as well as readings for dityrosine cross-linking and X-ray elemental microanalysis, suggesting a disruption in the secretory routes responsible for the chorion biogenesis. In addition, the ovaries from silenced insects presented altered levels of autophagy-related genes as well as a tendency of upregulation in ER chaperones, indicating a disturbance in the general biosynthetic-secretory pathway. Altogether, we found that E1 and E2 enzymes are essential for chorion biogenesis and that their silencing triggers the modulation of autophagy genes suggesting a coordinated function of both pathways for the progression of choriogenesis.

Comparative analysis of detoxification-related gene superfamilies across five hemipteran species.

BACKGROUND: Hemiptera is one of the most speciose orders of insects, and the most speciose considering Hemimetabola. Through their evolutive history, hemipterans with different feeding habits have adapted to deal with different chemical challenges. Three major gene families are involved in xenobiotic detoxification in insects: the cytochromes P450 (CYPs), carboxyl/cholinesterases (CCEs), and glutathione transferases (GSTs). Here we perform a comparative analysis on the complement of these gene superfamilies across five hemipteran species; four heteropterans (the pentatomid plant feeders Nezara viridula and Halyomorpha halys; the hematophagous Cimex lectularius, Cimicidae, and Rhodnius prolixus, Reduviidae), and one Auchenorrhyncha plant feeder (Nilaparvata lugens). RESULTS: Our results point to an expansion of several enzyme families associated with xenobiotic detoxification in heteropterans with respect to other species and the existence of a dynamic evolution pattern including CYP3 clan, hormone and pheromone processing class in the CCE superfamily, and sigma class in GST superfamily. Other detoxification-related families are reduced in the hemipteran species analyzed here: reduction or even absence of epsilon class and reduced delta class in GST superfamily; absence of mitochondrial CYP12 family; absence of CYP9 family in CYP3 clan; and reduction or even absence of some dietary/detoxification groups of CCEs. Interestingly, the most polyphagous species analyzed here (H. halys) is also the one that presents the largest repertoire of detoxification enzymes. Gene cluster analysis suggests that this could be due to gene duplication events. CONCLUSIONS: The evolutionary analysis performed here reveals characteristics that are both common and particular for heteropterans. The composition and organization of detoxification-related gene families could shed light on evolutionary forces that shaped their divergence. These families are important for both the detoxification of diet products and for conferring tolerance or resistance to synthetic insecticides. Furthermore, we present the first comprehensive analysis of detoxification gene superfamilies in N. viridula, an understudied species in spite of its economic relevance as a crop pest. The information obtained is of interest for basic insect science as well as for the control of harmful species and the management of insecticide resistance.

The transition from vitellogenesis to choriogenesis triggers the downregulation of the UPR sensors IRE1 and PERK and alterations in the ER architecture in the follicle cells of the vector Rhodnius prolixus.

In insects, the follicle cells (FCs) give rise to a single-layered tissue of binucleated professional secretory cells that surround the oocytes during oogenesis. In the latest stage of oocyte development, the FCs rapidly synthesize and secrete the chorion (eggshell) immediately before degenerating through apoptosis. Here, we used RT-qPCR, electron microscopy, and RNAi silencing to explore the role of the main unfolded protein response (UPR) receptors IRE1 and PERK, as well as the ultrastructure dynamics of the FCs during oogenesis of the insect vector of Chagas disease Rhodnius prolixus. We found that IRE1 and PERK mRNAs are highly expressed in the ovaries of vitellogenic females. Interestingly, we observed that IRE1 and PERK, as well as different isoforms of the chaperones Bip and PDI, have their FCs gene expression levels decreased during the vitellogenesis to choriogenesis transition. Using transmission electron microscopy, we observed that the downregulation of the UPR gene expression is accompanied by dramatic changes in the FCs ultrastructure, with an 80% reduction in the mean area of the ER tubules, and circularization and enlargement of the mitochondria. Additionally, we found that parental RNAi silencing of both IRE1 and PERK resulted in minor changes in the chorion protein composition and ultrastructure, accessed by urea extraction of the chorion proteins and scanning electron microscopy, respectively, but did not impact the overall levels of oviposition and F1 embryo development.

Benefits and costs of immune memory in Rhodnius prolixus against Trypanosoma cruzi.

There is increasing evidence supporting the immune memory in invertebrates, but the studies are relatively neglected in insect vectors other than mosquitoes. Therefore, we tested two hypotheses: 1) Rhodnius prolixus insects possess immune memory against Trypanosoma cruzi, and 2) their immune memory is costly. The Dm28c and Y strains of T. cruzi were used, the former being more infective than the latter. On the one hand, the triatomines subjected to dual challenges with the Dm28c strain did not show significant differences in survival than those of the heterologous challenge groups control-Dm28c and Y-Dm28c. On the other hand, the insects survived longer after a dual Y-Y challenge than after the corresponding heterologous challenge (control-Y). The Y-Y, Dm28c-Y, and naïve groups showed similar survival. There was more prolonged survival following the Y-Y versus Dm28c-Dm28c dual challenge. The Dm28c-Dm28c group exhibited moulting sooner than the control-Dm28c or naïve group. In contrast, there were no differences in the probability of moulting between the Y-Y and naïve groups. The results suggest that triatomines have immune memory against the Y but not the Dm28c strain. Further investigation on triatomine and T. cruzi interaction is needed to determine if infectivity accelerates or delay growth due to innate immune memory.

Effect of salivary CYP4EM1 and CYP4EM2 gene silencing on the life span of Chagas disease vector Rhodnius prolixus (Hemiptera, Reduviidae) exposed to sublethal dose of deltamethrin.

Control of Chagas disease in endemic countries is primarily accomplished through insecticide spraying for triatomine vectors. In this context, pyrethroids are the first-choice insecticide, and the evolution of insect resistance to these insecticides may represent an important barrier to triatomine control. In insects, cytochrome P450s are enzymes involved in the metabolism of xenobiotics and endogenous chemicals that are encoded by genes divided into different families. In this work, we evaluated the role of three Rhodnius prolixus CYP4EM subfamily genes during blood meal and after deltamethrin exposure. CYP4 gene members were expressed in different insect organs (integument, salivary glands (SGs), midgut, fat body and malpighian tubules) at distinct transcriptional levels. CYP4EM1 gene was highly expressed in the SG and was clearly modulated after insect blood meal. Injection of CYP4EM1dsRNA promoted significant reduction in mRNA levels of both CYP4EM1 and CYP4EM2 genes and induced deleterious effects in R. prolixus nymphs subsequently exposed to sublethal doses of deltamethrin (3.4 or 3.8 ng/nymph treated). The higher dose reduced the survival over time and increased susceptibility of R. prolixus nymphs to deltamethrin. A better understanding of this mechanism can help in developing of more efficient strategies to reduce Trypanosoma cruzi vector transmission in Americas.

The role of neuropeptides in regulating ecdysis and reproduction in the hemimetabolous insect Rhodnius prolixus.

In ecdysozoan animals, moulting entails the production of a new exoskeleton and shedding of the old one during ecdysis. It is induced by a pulse of ecdysone that regulates the expression of different hormonal receptors and activates a peptide-mediated signalling cascade. In Holometabola, the peptidergic cascade regulating ecdysis has been well described. However, very little functional information regarding the neuroendocrine regulation of ecdysis is available for Hemimetabola, which display an incomplete metamorphosis. We use Rhodnius prolixus as a convenient experimental model to test two hypotheses: (1) the role of neuropeptides that regulate ecdysis in Holometabola is conserved in hemimetabolous insects; and (2) the neuropeptides regulating ecdysis play a role in the regulation of female reproduction during the adult stage. The RNA interference-mediated reduction of ecdysis triggering hormone (ETH) mRNA levels in fourth-instar nymphs resulted in lethality at the expected time of ecdysis. Unlike in holometabolous insects, knockdown of eth and orcokinin isoform A (oka) did not affect oviposition in adult females, pointing to a different endocrine regulation of ovary maturation. However, eth knockdown prevented egg hatching. The blockage of egg hatching appears to be a consequence of embryonic ecdysis failure. Most of the first-instar nymphs hatched from the eggs laid by females injected with dsRNA for eclosion hormone (dsEH), crustacean cardioactive peptide (dsCCAP) and dsOKA died at the expected time of ecdysis, indicating the crucial involvement of these genes in post-embryonic development. No phenotypes were observed upon corazonin (cz) knockdown in nymphs or adult females. The results are relevant for evolutionary entomology and could reveal targets for neuropeptide-based pest control tools.