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.

The Rpfor gene modulates the locomotory activity and host-seeking behaviour of Rhodnius prolixus.

The molecular bases of animal behaviour are intricate due to the pleiotropic nature of behaviour-modulating genes, which are often expressed across multiple tissues. The foraging gene (for) encodes a cGMP-dependent protein kinase (PKG), pivotal in regulating downstream target proteins through phosphorylation. In insects, for has been implicated in various behavioural contexts and physiological processes regarding searching for food. Rhodnius prolixus, a hematophagous bug that transmits Trypanosoma cruzi, the causative agent of Chagas disease, exhibits specific activity patterns associated with its hematophagous behaviour. Our previous work demonstrated a correlation between locomotor activity profiles and the expression of Rpfor, suggesting its involvement in modulating triatomine locomotion. In this study, we investigated the impact of Rpfor knockdown on locomotory activity, host-seeking behaviour, feeding performance and lipid metabolism in R. prolixus nymphs. Using RNA interference, we achieved a significant reduction of Rpfor expression in both the brain and fat body of R. prolixus nymphs. Knocked-down nymphs exhibited diminished non-oriented locomotory activity compared with controls, without altering the characteristic bimodal pattern of activity. Additionally, they displayed an increased tendency to approach a host, suggesting a role for Rpfor in modulating host-seeking behaviour. Feeding performance and lipid metabolism remained unaffected by Rpfor knockdown. Our findings underscore the multifaceted role of Rpfor in modulating locomotor activity and host-seeking behaviour in R. prolixus nymphs, shedding light on the molecular mechanisms underlying their hematophagous behaviour and potential implications for disease transmission. Further research is necessary to elucidate the intricate interplay between Rpfor expression, behaviour and physiological processes in triatomine bugs.

As bases moleculares do comportamento animal são complexas devido à natureza pleiotrópica dos genes envolvidos na sua modulação, normalmente expressos em múltiplos tecidos. O gene foraging (for) codifica para uma proteína quinase dependente de cGMP, fundamental para a regulação de proteínas alvo via fosforilação. Em insetos, o gene for tem sido associado a vários contextos comportamentais e processos fisiológicos relacionados com forrageamento. Rhodnius prolixus, um inseto hematófago que transmite Trypanosoma cruzi, o agente causativo da doença de Chagas, exibe padrões de atividade específicos associados com o seu comportamento hematófago. Em um estudo anterior, demonstramos uma correlação entre os perfis de atividade locomotora e a expressão de Rpfor, sugerindo o seu envolvimento na modulação da locomoção de triatomíneos. No presente estudo, investigamos o impacto do silenciamento de Rpfor na atividade locomotora, no comportamento de busca por hospedeiro, na performance alimentar, e no metabolismo de lipídeos em ninfas de R. prolixus. Através da técnica de RNA de interferência, obtivemos uma redução significativa da expressão do gene Rpfor no cérebro e no corpo gorduroso de R. prolixus. Insetos silenciados exibiram uma redução da atividade locomotora não orientada em comparação com controles, sem alterações no padrão bimodal da atividade. Adicionalmente, os insetos apresentaram um aumento no comportamento de busca por hospedeiro, sugerindo um papel para o Rpfor na sua modulação. A performance alimentar e o metabolismo de lipídeos não foram alterados pelo silenciamento do gene. Nossas descobertas ressaltam o papel multifuncional do gene Rpfor na modulação da atividade locomotora e no comportamento de busca por hospedeiro em R. prolixus, ampliando o conhecimento sobre os mecanismos moleculares relacionados ao seu comportamento hematófago e potenciais implicações para a transmissão de doenças. Estudos adicionais são necessários para elucidar a intrincada interação entre expressão, comportamento e processos fisiológicos de Rpfor em insetos triatomíneos.

Mechanisms controlling haemolymph circulation under resting conditions in the Chagas disease vector Rhodnius prolixus.

Chagas disease vectors can ingest several times their own volume in blood with each meal. This ad libitum feeding causes an intense process of diuresis, inducing the insect to eliminate a large quantity of urine and faeces. To ensure diuresis, the speed of circulation of the haemolymph is increased. The Triatominae circulatory system is quite simple, including the dorsal vessel, which pumps haemolymph in an anterograde direction. The return is caused by peristaltic contractions of the anterior midgut. Triatominae insects can spend several weeks without feeding, meaning that most of the time, the insect is in a resting condition. Although the mechanisms controlling the circulation of the haemolymph during post-prandial diuresis have been largely analysed, the mechanisms controlling it during resting conditions are poorly understood. In this study, we analysed several canonical pathways (i.e. L-type VGCC, GPCR, RyR, IP3R) and a novel system represented by the recently characterized Piezo proteins. Our results show that during the resting condition, haemolymph circulation depends on a cross-talk between myogenic activity, inhibitory and stimulatory cellular messengers, and Piezo proteins. This report also unveils for the first time the existence of a putative Piezo protein in Hemiptera.

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.

What happens when the insecticide does not kill? A review of sublethal toxicology and insecticide resistance in triatomines.

Chagas disease is considered one of the most important human parasitosis in the United States. This disease is mainly transmitted by insects of the subfamily Triatominae. The chemical vector control is the main tool for reducing the incidence of the disease. However, the presence of triatomines after pyrethroids spraying has been reported in some regions, as in the case of Triatoma infestans in Argentina and Bolivia. The presence of insects can be explained by the colonization from neighbouring areas, the reduction of insecticide dose to sublethal levels due to environmental factors, and/or by the evolution of insecticide resistance. In the last two scenarios, a proportion of the insects is not killed by insecticide and gives rise to residual populations. This article focuses on the toxicological processes associated with these scenarios in triatomines. Sublethal doses may have different effects on insect biology, that is, sublethal effects, which may contribute to the control. In addition, for insect disease vectors, sublethal doses could have negative effects on disease transmission. The study of sublethal effects in triatomines has focused primarily on the sequence of symptoms associated with nervous intoxication. However, the effects of sublethal doses on excretion, reproduction and morphology have also been studied. Rhodnius prolixus and T. infestans and pyrethroids insecticides were the triatomine species and insecticides, respectively, mainly studied. Insecticide resistance is an evolutionary phenomenon in which the insecticide acts as a selective force, concentrating on the insect population's pre-existing traits that confer resistance. This leads to a reduction in the susceptibility to the insecticide, which was previously effective in controlling this species. The evolution of resistance in triatomines received little attention before the 2000s, but after the detection of the first focus of resistance associated with chemical control failures in T. infestans from Argentina in 2002, the study of resistance increased remarkably. A significant number of works have studied the geographical distribution, the resistance mechanisms, the biological modifications associated with resistance, the environmental influences and the genetic of T. infestans resistant to pyrethroid insecticides. Currently, studies of insecticide resistance are gradually being extended to other areas and other species. The aim of this article was to review the knowledge on both phenomena (sublethal effects and insecticide resistance) in triatomines. For a better understanding of this article, some concepts and processes related to insect-insecticide interactions, individual and population toxicology and evolutionary biology are briefly reviewed. Finally, possible future lines of research in triatomine toxicology are discussed.

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.

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.

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.

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.

Trypanosoma rangeli infection impairs reproductive success of Rhodnius prolixus.

Trypanosoma rangeli is a protozoan that infects triatomines and mammals in Central and South America. Although it does not cause disease to humans, this parasite produces different levels of pathogenicity to its invertebrate host, mainly in species of the genus Rhodnius. In this study, we followed T. rangeli-infected and uninfected pairs throughout their adult lives and measured the amount of blood ingested, number of eggs laid, number of eggs hatched and proportion of infertile eggs, as well as female life expectancy. We found that all reproductive parameters were drastically decreased during infection, mainly due to the reduced amount of blood the infected insects ingested throughout their lives. Reproductive parameters were also affected by the reduction of the life expectancy of infected females, as survival was positively correlated with the number of eggs laid. The strategies used by the parasite to be transmitted are discussed in view of the pathological effects it causes in the insect.

Trypanosoma rangeli infection increases the exposure and predation endured by Rhodnius prolixus.

Trypanosoma rangeli is a protozoan that infects triatomines and mammals in Latin America, sharing hosts with Trypanosoma cruzi, the etiological agent of Chagas disease. Trypanosoma rangeli does not cause disease to humans but is strongly pathogenic to its invertebrate hosts, increasing mortality rates and affecting bug development and reproductive success. We have previously shown that this parasite is also capable of inducing a general increase in the locomotory activity of its vector Rhodnius prolixus in the absence of host cues. In this work, we have evaluated whether infection impacts the insect­vertebrate host interaction. For this, T. rangeli-infected and uninfected R. prolixus nymphs were released in glass arenas offering single shelters. After a 3-day acclimatization, a caged mouse was introduced in each arena and shelter use and predation rates were evaluated. Trypanosoma rangeli infection affected all parameters analysed. A larger number of infected bugs was found outside shelters, both in the absence and presence of a host. Infected bugs also endured greater predation rates, probably because of an increased number of individuals that attempted to feed. Interestingly, mice that predated on infected bugs did not develop T. rangeli infection, suggesting that the oral route is not effective for these parasites, at least in our system. Finally, a smaller number of infected bugs succeeded in feeding in this context. We suggest that, although T. rangeli is not transmitted orally, an increase in the proportion of foraging individuals would promote greater parasite transmission rates through an increased frequency of very effective infected-bug bites.

Ecdysone modulates both ultrastructural arrangement of hindgut and attachment of Trypanosoma cruzi DM 28c to the rectum cuticle of Rhodnius prolixus fifth-instar nymph.

Studies on the effects of azadirachtin treatment, ecdysone supplementation and ecdysone therapy on both the ultrastructural organization of the rectum in 5th-instar nymph of Rhodnius prolixus and the ex vivo attachment behavior of Trypanosoma cruzi under these experimental conditions were carried out. Control insects had a typical and significant organization of the rectum cuticle consisted of four main layers (procuticle, inner epicuticle, outer epicuticle, and wax layer) during the entire period of the experiment. Both azadirachtin treatment and ecdysone supplementation avoid the development of both outer epicuticle and wax layer. Oral therapy with ecdysone partially reversed the altered organization and induce the development of the four main rectal cuticle layers. In the same way, the ex vivo attachment of T. cruzi to rectal cuticle was blocked by azadirachtin treatment but ecdysone therapy also partially recovered the parasite adhesion rates to almost those detected in control insects. These results point out that ecdysone may be a factor responsible - directly or indirectly - by the modulation of rectum ultrastructural arrangement providing a superficial wax layer to the attachment followed by metacyclogenesis of T. cruzi in the rectum of its invertebrate hosts.

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.

The neuropeptide RhoprCCHamide2 inhibits serotonin-stimulated transcellular Na+ transport across the anterior midgut of the vector of Chagas disease, Rhodnius prolixus.

Rhodnius prolixus is a blood-feeding insect vector of Trypanosoma cruzi, a protozoan parasite that causes Chagas disease. During each blood meal, the animals ingest large volumes of blood, that may be up to 12 times the unfed body mass. These blood meals impose a significant osmotic stress for the animals due to the hyposmotic condition of the ingested blood compared with the insect's hemolymph. Thus the insect undergoes a massive postprandial diuresis that allows for the excretion of the plasma fraction of the blood in less than two hours. Diuresis is performed by the excretory system, consisting of the Malpighian tubules and gut, under the control of diuretic and anti-diuretic factors. We investigated the ion transport machinery triggered by stimulation with the diuretic factor serotonin in the anterior midgut (i.e. crop) and the effect of the diuretic modulator RhoprCCHamide2. Ussing chamber assays revealed that serotonin-stimulated increase in transepithelial short-circuit current (Isc) was more sensitive to the blockage with amiloride than 5-N-ethyl-N-isopropyl amiloride (EIPA), suggesting the involvement of Na+ channels. Incubation in Na+-free, but not Cl--free saline, blocked the effect of serotonin on Isc. Moreover, treatment with Na+-K+-2Cl- cotransporter (NKCC) and Na+-Cl- cotransporter (NCC) blockers had no effect on fluid secretion but was blocked by amiloride. Blockage of Na+/K+-ATPase with ouabain inhibited Isc but the H+-ATPase inhibitor bafilomycin had no effect. The neuropeptide RhoprCCHamide2 diminished serotonin-stimulated Isc across the crop. The results suggest that Na+ undergoes active transport via an apical amiloride-sensitive Na+ channel and a basolateral ouabain-sensitive Na+/K+-ATPase, while Cl- is transported through a passive paracellular pathway.

Bioecological aspects of triatomines and marsupials as wild Trypanosoma cruzi reservoirs in urban, peri-urban and rural areas in the Western Brazilian Amazon.

In the Amazon region, Trypanosoma cruzi transmission cycles involve a great diversity of Triatominae vectors and mammal reservoirs. Some Rhodnius spp. mainly inhabit palm trees that act as microhabitats for hosts and vectors. The current study aimed to describe aspects of the bio-ecology of the vectors and reservoirs of T. cruzi in relation to human populations resident near areas with large quantities of palm trees, in rural, peri-urban and urban collection environments, located in the Western Brazilian Amazon. Rhodnius pictipes and Didelphis marsupialis were respectively the most predominant vector and reservoir, with rates of 71% for R. pictipes and 96.5% for D. marsupialis. The vast majority of T. cruzi isolates clustered with TcI. The most prevalent haplotype was TcI COII1 (69.7%). Mauritia flexuosa and Attalea phalerata were the main ecological indicators of infestation by triatomines. Birds were the most common food source (27,71%). T. cruzi isolated from R. robustus has the haplotype HUM-13, previously detected in a chronic Chagas patient living in the same area. Our results demonstrate the relevance of this study, with the occurrence of elevated infection rates in animals, and suggest the importance of the Amazon zones where there is a risk of infection in humans.

Satellitome Analysis of Rhodnius prolixus, One of the Main Chagas Disease Vector Species.

The triatomine Rhodnius prolixus is the main vector of Chagas disease in countries such as Colombia and Venezuela, and the first kissing bug whose genome has been sequenced and assembled. In the repetitive genome fraction (repeatome) of this species, the transposable elements represented 19% of R. prolixus genome, being mostly DNA transposon (Class II elements). However, scarce information has been published regarding another important repeated DNA fraction, the satellite DNA (satDNA), or satellitome. Here, we offer, for the first time, extended data about satellite DNA families in the R. prolixus genome using bioinformatics pipeline based on low-coverage sequencing data. The satellitome of R. prolixus represents 8% of the total genome and it is composed by 39 satDNA families, including four satDNA families that are shared with Triatoma infestans, as well as telomeric (TTAGG)n and (GATA)n repeats, also present in the T. infestans genome. Only three of them exceed 1% of the genome. Chromosomal hybridization with these satDNA probes showed dispersed signals over the euchromatin of all chromosomes, both in autosomes and sex chromosomes. Moreover, clustering analysis revealed that most abundant satDNA families configured several superclusters, indicating that R. prolixus satellitome is complex and that the four most abundant satDNA families are composed by different subfamilies. Additionally, transcription of satDNA families was analyzed in different tissues, showing that 33 out of 39 satDNA families are transcribed in four different patterns of expression across samples.

Sensory discrimination between aversive salty and bitter tastes in an haematophagous insect.

Sensory aversion is essential for avoiding prospective dangers. We studied the chemical perception of aversive compounds of different gustatory modalities (salty, bitter) in the haematophagous bug, Rhodnius prolixus. Over a walking arena, insects avoided a substrate embedded with 1M NaCl or KCl if provided with water as an alternative. However, no preferences were expressed when both salts were opposed to each other. A pre-exposure to amiloride interfered with the repellency of NaCl and KCl equally, suggesting that amiloride-sensitive receptors are involved in the detection of both salts. Discriminative experiments were then performed to determine whether R. prolixus can distinguish between these salts. An aversive operant conditioning involving either NaCl or KCl modulated the repellency of the conditioned salt, but also of the novel salt. Repellency levels of both salts were rigid to a chemical pre-exposure to any of both salts. When gustatory modalities were crossed by presenting as a choice NaCl and a bitter molecule as caffeine (Caf), no innate preferences were expressed. Aversive operant conditionings with either NaCl or Caf rendered unspecific changes in the repellency of both compounds. A chemical pre-exposure to Caf modulated the response to Caf but not to NaCl, suggesting the existence of two independent neural pathways for the detection of salts and bitter compounds. Overall results suggest that R. prolixus cannot discriminate molecules of the same gustatory modality (i.e. salty), but can distinguish between salty and bitter tastes. The potential use of aversive gustatory stimuli as a complement of commercially available olfactory repellents is discussed.

Rhodnius spp. are differentiated based on the peptide/protein profile by matrix-assisted laser desorption/ionization mass spectrometry and chemometric tools.

Triatominae are hematophagous insects involved in the transmission of Chagas disease. Among the 19 genera of the subfamily, those with the highest epidemiological importance regarding the dissemination of Trypanosoma cruzi are Panstrongylus, Rhodnius, and Triatoma. Of these three genera, Rhodnius presents the greatest difficulties for specific identification. Thus, there is a need to overcome the difficulties in identifying phenotypes of similar species of this genus. In the present study, the MALDI-TOF MS methodology was used to identify 12 Rhodnius species, among the 21 admitted. The MALDI-TOF MS methodology allowed specific characterization through the identification of peptides and proteins, starting from four different methods of extraction: (A) acetonitrile/formic acid (ACN/AF), (B) acetonitrile/trifluoroacetic acid (ACN/TFA), (C) isopropyl/formic acid (IPA/AF), and (D) methanol/formic acid (MeOH/AF), and four types of MALDI-TOF matrices: α-cyano-4-hydroxycinnamic acid (CHCA), sinapic acid (SA), 6-aza-2-thiothymine (ATT), and 2,6-dihydroxyacetophenone (DHAP). The experiments were performed by combining the four solvents and four matrices to select the best MALDI extraction/matrix. The application of the MALDI-TOF MS technique, through the digital mass spectrometry approach combined with chemometric tools, such as partial least squares-discriminant analysis (PLS-DA), was able to discriminate 12 species of Rhodnius genus, which are difficult to identify using morphological characteristics. Thus, in view of the results obtained, the methodology described in the present article can be applied with speed and efficiency for the discrimination of Triatominae species. Graphical Abstract.

Triatomines of the Genus Rhodnius Do Not Mark Shelters with Feces.

Aggregation to volatile compounds emitted by feces has been demonstrated for several triatomine species. This signal guides the insects to suitable places that offer physical protection while providing contact with conspecifics. Though the use of aggregation marks has also been reported for Rhodnius spp., it is still unclear whether feces really cause these insects to aggregate inside shelters. In two-choice assays using artificial shelters, we found that refuges associated with a blend of synthetic compounds based on volatiles released by the feces of some triatomine species and reported to be attractive to several species, did not induce shelter choice in Rhodnius prolixus Stål, 1859. In addition, we show that refuges associated with feces of conspecifics did not induce shelter choice in R. prolixus, R. robustus Larrousse, 1927, R. neglectus Lent, 1954, and R. ecuadoriensis Lent and León, 1958. In contrast, as expected, control experiments with Triatoma infestans (Klug, 1834), another triatomine known to aggregate in shelters marked with feces, definitely showed preference for shelters containing feces of conspecifics. Our results clearly show that volatile signals associated with feces do not mediate shelter choice in Rhodnius spp. As a consequence, a paradigm shift will be necessary and, consequently, ab ovo investigations on the clues inducing aggregation behavior in these species.