Long-term effects and parental RNAi in the blood feeder Rhodnius prolixus (Hemiptera; Reduviidae)

RNA interference (RNAi) has been widely employed as a useful alternative to study gene function in insects, including triatomine bugs. However, several aspects related to the RNAi mechanism and functioning are still unclear. The aim of this study is to investigate the persistence and the occurrence of systemic and parental RNAi in the triatomine bug Rhodnius prolixus. For such, the nitrophorins 1 to 4 (NP1-4), which are salivary hemeproteins, and the rhodniin, an intestinal protein, were used as targets for RNAi. The dsRNA for both molecules were injected separately into 3rd and 5th instar nymphs of R. prolixus and the knockdown (mRNA levels and phenotype) were progressively evaluated along several stages of the insect's life. We observed that the NP1-4 knockdown persisted for more than 7 months after the dsRNA injection, and at least 5 months in rhodniin knockdown, passing through various nymphal stages until the adult stage, without continuous input of dsRNA. The parental RNAi was successful from the dsRNA injection in 5th instar nymphs for both knockdown targets, when the RNAi effects (mRNA levels and phenotype) were observed at least in the 2nd instar nymphs of the F1 generation. However, the parental RNAi did not occur when the dsRNA was injected in the 3rd instars. The confirmation of the long persistence and parental transmission of RNAi in R. prolixus can improve and facilitate the utilization of this tool in insect functional genomic studies.     

Sn-protoporphyrin inhibits both heme degradation and hemozoin formation in Rhodnius prolixus midgut

Hematophagy is a feeding habit that involves the ingestion of huge amounts of heme. The hematophagous hemipteran Rhodnius prolixus evolved many genetic resources to protect cells against heme toxicity. The primary barrier against the deleterious effects of heme is the aggregation of heme into hemozoin in the midgut lumen. Hemozoin formation is followed by the enzymatic degradation of heme by means of a unique pathway whose end product is dicysteinyl-biliverdin IX-γ (Rhodnius prolixus biliverdin, RpBv). These mechanisms are complemented by a heme-binding protein (RHBP) in the hemolymph that attenuates the pro-oxidant effects of heme. In this work, we show that when insects are fed with blood enriched with a heme analog, Sn-protoporphyrin (SnPP-IX), both hemozoin synthesis and RpBv production are inhibited in a dose-dependent manner. These effects are accompanied by increased oxidative damage to the midgut epithelium and inhibition of oviposition, indicating that hemozoin formation and heme degradation are protective mechanisms that work together and contributed to the adaptation of this insect to successfully feed on vertebrate blood.     

Silencing of Maternal Heme-binding Protein Causes Embryonic Mitochondrial Dysfunction and Impairs Embryogenesis in the Blood Sucking Insect Rhodnius prolixus

The heme molecule is the prosthetic group of many hemeproteins involved in essential physiological processes, such as electron transfer, transport of gases, signal transduction, and gene expression modulation. However, heme is a pro-oxidant molecule capable of propagating reactions leading to the generation of reactive oxygen species. The blood-feeding insect Rhodnius prolixus releases enormous amounts of heme during host blood digestion in the midgut lumen when it is exposed to a physiological oxidative challenge. Additionally, this organism produces a hemolymphatic heme-binding protein (RHBP) that transports heme to pericardial cells for detoxification and to growing oocytes for yolk granules and as a source of heme for embryo development. Here, we show that silencing of RHBP expression in female fat bodies reduced total RHBP circulating in the hemolymph, promoting oxidative damage to hemolymphatic proteins. Moreover, RHBP knockdown did not cause reduction in oviposition but led to the production of heme-depleted eggs (white eggs). A lack of RHBP did not alter oocyte fecundation. However, produced white eggs were nonviable. Embryo development cellularization and vitellin yolk protein degradation, processes that normally occur in early stages of embryogenesis, were compromised in white eggs. Total cytochrome c content, cytochrome c oxidase activity, citrate synthase activity, and oxygen consumption, parameters that indicate mitochondrial function, were significantly reduced in white eggs compared with normal dark red eggs. Our results showed that reduction of heme transport from females to growing oocytes by RHBP leads to embryonic mitochondrial dysfunction and impaired embryogenesis.     

Symbiont-mediated RNA interference in insects

RNA interference (RNAi) methods for insects are often limited by problems with double-stranded (ds) RNA delivery, which restricts reverse genetics studies and the development of RNAi-based biocides. We therefore delegated to insect symbiotic bacteria the task of: (i) constitutive dsRNA synthesis and (ii) trauma-free delivery. RNaseIII-deficient, dsRNA-expressing bacterial strains were created from the symbionts of two very diverse pest species: a long-lived blood-sucking bug, Rhodnius prolixus, and a short-lived globally invasive polyphagous agricultural pest, western flower thrips (Frankliniella occidentalis). When ingested, the manipulated bacteria colonized the insects, successfully competed with the wild-type microflora, and sustainably mediated systemic knockdown phenotypes that were horizontally transmissible. This represents a significant advance in the ability to deliver RNAi, potentially to a large range of non-model insects.     

Uptake of Rhodnius heme-binding protein (RHBP) by the ovary of Rhodnius prolixus

The uptake of RHBP (Rhodnius heme-binding protein) by the ovaries of Rhodnius prolixus was characterized. RHBP purified from oocyte was labeled with ¹²⁵I and used to study the process of uptake by the ovary in vivo and in vitro. After injection, the [¹²⁵I]RHBP was readily removed from the hemolymph and accumulated especially in the ovary. The capacity of the ovary to take up [¹²⁵I]RHBP from the hemolymph varied during the days following blood meal. It increased up to day 2, remained stable until day 5, and then decreased up to the end of oogenesis. In vitro, the uptake of [¹²⁵I]RHBP was linear at least up to 60 min. The uptake was dependent on [¹²⁵I]RHBP concentration and showed to be a saturable process. The addition of a molar excess of non-related proteins such as Vitellin (Vt), Lipophorin (Lp), and Bovine Serum Albumin (BSA) did not reduce [¹²⁵I]RHBP uptake. Using immunogold technique the RHBP was localized at the microvilli, coated pits, and yolk granules. The main yolk protein, Vt, did not compete with RHBP for the uptake. Thus, it is discussed here that they bind to independent binding sites of the oocytes, and are directed later on to the same compartment. The need of both proteins for the completion of mature oocyte was verified in vivo. The reduction of heme-RHBP in the hemolymph, by changing the diet, decreased the number of eggs laid. Increasing the concentration of heme-RHBP in the hemolymph, the number of eggs produced increased in a dose dependent manner. In vitro, both apo-RHBP and heme-RHBP can be taken up by the oocyte. Since the mature oocyte contains only heme-saturated RHBP, the possible fate of apo-RHBP is also discussed. Arch. Insect Biochem. Physiol. 39:133–143, 1998. © 1998 Wiley-Liss, Inc.     

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.     

Amines from vertebrates guide triatomine bugs to resources

Most triatomine bugs (Heteroptera: Reduviidae) are nest-living insects that require vertebrate blood or invertebrate haemolymph to complete their life cycle. Vertebrates accumulate excretory products in or near their nesting sites and we hypothesize that triatomines use emanations from such host wastes when searching for resources. Here we recount how triatomine bugs increase upwind locomotion on a servosphere in response to volatile amine constituents of vertebrate excretions. Fresh chicken faeces is strongly attractive to Rhodnius prolixus nymphs. Ammonia induces attraction and an increase in both speed and total path length by R. prolixus on the servosphere. Whereas ethylamine and dimethylamine attract R. prolixus, Triatoma infestans and Panstrongylus geniculatus, other amine constituents of vertebrate excretions such as isobutylamine and hexylamine induce R. prolixus nymphs to walk faster and for a longer period. These amines are derived from generally occurring metabolites of vertebrates and from gut flora metabolism. We conclude that amines and other products associated with nesting hosts serve as signals for foraging triatomines.     

Influence of the intestinal anticoagulant in the feeding performance of triatomine bugs (Hemiptera; Reduviidae)

Triatomines are haematophagous insects in all post-embryonic life stages. They are vectors of Trypanosoma cruzi, the causative agent of Chagas disease. Their vectorial ability is influenced by their feeding performance, which varies greatly amongst species. Recent work showed that inhibition of the coagulation process in the anterior midgut (crop) environment considerably influences the blood meal size. In this work, we performed a comparative study of the level of anticoagulant activity in the saliva and crop contents of three triatomine species - Triatoma infestans, Triatoma brasiliensis and Rhodnius prolixus - and correlated this with their feeding performance on live hosts. Moreover, the feeding parameters on a large diameter vessel influenced by the crop anticoagulants were evaluated in detail. The anticoagulant activity was significantly higher in the crop contents than in salivary glands, varying from 1.6-fold higher for R. prolixus to 70-fold higher for T. brasiliensis. Amongst the species, T. brasiliensis had the lowest crop anticoagulant activity, the lowest concentration of thrombin inhibitor, and took the longest to feed. Triatoma brasiliensis nymphs that had their intestinal anticoagulant (brasiliensin) knocked down by RNA interference had the lowest capacity to maintain cibarial pump frequency at higher levels throughout the feeding process and consequently a lower ingestion rate (mg/min), even when fed under favourable conditions (large diameter vessel). However, the feeding difficulty for brasiliensin knockdown T. brasiliensis nymphs was reversed by treating the host mice with heparin (a potent systemic anticoagulant) before blood feeding. The results indicate that crop anticoagulant activity influences modulation of the blood-pumping frequency to the intestine and significantly affects the feeding efficiency of triatomine spp. on live hosts.     

Protein 2DE reference map of the anterior midgut of the blood‐sucking bug Rhodnius prolixus

Rhodnius prolixus is an important vector of Trypanosoma cruzi, the causative agent of Chagas’ disease, an illness that affects 20% of Latin America population. The obligatory course of the parasite in the vector digestive tract has made it an important target for investigation in order to control the parasite transmission and thus interrupt its biological cycle in the insect vector. Therefore, an insight into the vector midgut physiology is valuable for insect control as well as to provide potential novel targets for drugs and vaccines development and thus disease treatment. In this study, the first 2DE map of R. prolixus anterior midgut is described. Proteins were separated by 2DE and analyzed by nano‐LC MS/MS. The results yielded 489 proteins from 475 spots. These proteins were classified into 28 functional groups and their physiological roles in the insect midgut are discussed. All MS data have been deposited in the ProteomeXchange with identifiers PXD001488 and PXD001489 ( http://proteomecentral.proteomexchange.org/dataset/PXD001488 , http://proteomecentral.proteomexchange.org/dataset/PXD001489 ).     

Growth retardation of Rhodnius prolixus symbionts by immunizing host against Nocardia (Rhodococcus) rhodnii

The possiblity of suppressing the growth of Nocardia (Rhodococcus) rhodnii symbiont in the gut of Rhodnius prolixus by feeding the bugs on rabbits immunized against the bacterium, was investigated. Rabbits immunized with symbionts isolated from adult bugs and grown in vitro developed high antisymbiont titres (1,024-2,048) and showed no ill side effects. The log-phase growth of the symbionts was retarded in the gut of R. prolixus larvae fed on immunized rabbits. Both growth rate and maximal number of symbionts were lower in groups receiving treatment. Rate of development of bugs reared on immunized rabbits was slowed and their success in moulting was reduced.     

Knockdown of a putative Halloween gene Shade reveals its role in ecdysteroidogenesis in the small brown planthopper Laodelphax striatellus

Ecdysteroid hormone 20-hydroxyecdysone (20E) plays fundamental roles in insect development and reproduction, whereas the primary role of ecdysone (E) is the precursor for 20E. A cytochrome P450 monooxygenase (CYP), encoded by a Halloween gene Shade (Shd, cyp314a1), catalyzes the conversion of E into 20E in representative insect species in Diptera, Lepidoptera and Orthoptera. We describe here the cloning and characterization of LsShd in a hemipteran insect species, the small brown planthopper Laodelphax striatellus. LsSHD has five insect conserved P450 motifs, i.e., Helix-C, Helix-I, Helix-K, PERF and heme-binding motifs. Temporal expression pattern of LsShd was determined through the fourth-instar and the early fifth-instar stages by qPCR. LsShd showed two expression peaks in day 2 and day 5 fourth-instar nymphs, and two troughs in day 1 fourth and fifth instars. Dietary introduction of double-stranded RNA (dsRNA) of LsShd into nymphs successfully knocked down the target gene, decreased expression level of ecdysone receptor (LsEcR) gene, and caused nymphal lethality and delayed development. Ingestion of 20E did not increase LsShd expression level, but almost completely rescued LsEcR mRNA level, and relieved the negative effects on the survival and development in LsShd-dsRNA-exposed nymphs. In contrast, dietary introduction of E had little rescue effects. Thus, our data suggest that the ecdysteroidogenic pathway is conserved in insects, and LsSHD functions to regulate metamorphotic processes by converting E to 20E even in a hemipteran insect, L. striatellus.     

CALCIUM‐INDUCED LIPID PEROXIDATION IS MEDIATED BY RHODNIUS HEME‐BINDING PROTEIN (RHBP) AND PREVENTED BY VITELLIN

Lipid peroxidation is promoted by the quasi‐lipoxygenase (QL) activity of heme proteins and enhanced by the presence of free calcium. Unlike mammalian plasma, the hemolymph of Rhodnius prolixus, a vector of Chagas disease, contains both a free heme‐binding protein (RHBP) and circulating lipoproteins. RHBP binds and prevents the heme groups of the proteins from participating in lipid peroxidation reactions. Herein, we show that despite being bound to RHBP, heme groups promote lipid peroxidation through a calcium‐dependent QL reaction. This reaction is readily inhibited by the presence of ethylene glycol tetraacetic acid (EGTA), the antioxidant butylated hydroxytoluene or micromolar levels of the main yolk phosphoprotein vitellin (Vt). The inhibition of lipid peroxidation is eliminated by the in vitro dephosphorylation of Vt, indicating that this reaction depends on the interaction of free calcium ions with negatively charged phosphoamino acids. Our results demonstrate that calcium chelation mediated by phosphoproteins occurs via an antioxidant mechanism that protects living organisms from lipid peroxidation.