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In triatomines, blood-feeding triggers many physiological processes including post-embryonic development and reproduction. Different feeding habits, such as hematophagy, can shape gene functions to meet the challenges of each type of diet. The gut of blood-sucking insects faces particular challenges after feeding due to the quantity and the quality of the food ingested. A comparison of transcriptomic and proteomic data indicates that post-transcriptional regulation of gene expression is crucial in the triatomine gut. It was proposed that eukaryotic translation initiation factor 3 subunit m (eIF3m) and eIF3e define two different eIF3 complexes with a distinct affinity for the different mRNAs, thus selecting the set of mRNAs to be translated and constituting a post-transcriptional mode of regulation of gene expression. Because the eIF3m is mainly expressed in the gut, we evaluated its relevance in Rhodnius prolixus physiology through RNA interference-mediated gene silencing. The knockdown of eIF3m reduced the digestion rate, affecting the processes triggered by a blood meal. Its silencing inhibited moulting and caused premature death in nymphs while impaired ovary development, oviposition and increased resistance to starvation in adult females. The survival of males after feeding (resistance to starvation) was not affected by eIF3m knockdown. The information regarding the eIF3m function in insects is scarce and the phenotypes observed in R. prolixus upon eIF3m silencing are different and more severe than those previously described in Drosophila melanogaster, indicating a pleiotropic role of this gene in triatomines. This article is protected by copyright. All rights reserved.

The use of chemical insecticides is the main control method for Blattella germanica worldwide. The prolonged and frequent use of insecticides produced the selection of insecticide-resistant individuals. The German cockroach is one of the most widespread urban pests in Argentina. In the last decades, resistance monitoring studies in this country demonstrated that there is a high prevalence of pyrethroid-resistant populations of B. germanica in the field. In this work, we studied the resistance mechanisms of a field-collected strain of B. germanica at toxicological, enzymatic, and molecular levels. A resistance ratio of 100 was obtained for the resistant strain when it was exposed to β-cypermethrin. The pretreatment with specific synergists (piperonyl butoxide and triphenyl phosphate) led to a significant increase in the toxicity of the pyrethroid, suggesting an involvement of oxidases and esterases in the detoxification of this insecticide. Moreover, esterase and oxidase activities in the resistant strain were 1.5-fold and 2-fold higher respectively, compared to the susceptible individuals. On the other hand, the voltage-gated sodium channel gene of the resistant cockroaches did not show nucleotidic substitutions in the domain II which are associated to knockdown resistance in this species. These results suggest that the main mechanism of resistance of the studied cockroaches' strain is metabolic, mainly due to an increase in the activity of oxidase and esterase enzymes. The results of this work in addition to other reports found in literature show that the extended use of a single active principle for cockroach control promotes the development of resistance leading to control failure in the field. In contrast, integrated pest management strategies include the use of different control tools in addition to chemical insecticides, which delay the appearance of resistance increasing the efficacy of pest control.

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.

Blood-sucking insects incorporate many times their body weight of blood in a single meal. As proteins are the major component of vertebrate blood, its digestion in the gut of hematophagous insects generates extremely high concentrations of free amino acids. Previous reports showed that the tyrosine degradation pathway plays an essential role in adapting these animals to blood feeding. Inhibiting 4-hydroxyphenylpyruvate dioxygenase (HPPD), the rate-limiting step of tyrosine degradation, results in the death of insects after a blood meal. Therefore, it was suggested that compounds that block the catabolism of tyrosine could act selectively on blood-feeding insects. Here we have evaluated the toxicity against mosquitoes of three HPPD inhibitors currently used as herbicides and in human health. Among the compounds tested, nitisinone (NTBC) proved to be more potent than mesotrione (MES) and isoxaflutole (IFT) in Aedes aegypti. NTBC was lethal to Ae. aegypti in artificial feeding assays (LD50: 4.36 µM), as well as in topical application (LD50: 0.0033 nmol/mosquito). NTBC was also lethal to Ae. aegypti populations that were resistant to neurotoxic insecticides, and it was lethal to other mosquito species (Anopheles and Culex). Therefore, HPPD inhibitors, particularly NTBC, represent promising new drugs for mosquito control. Since they only affect blood-feeding organisms, they would represent a safer and more environmentally friendly alternative to conventional neurotoxic insecticides., Centro Regional de Estudios Genómicos

The use of chemical insecticides is the main control method for Blattella germanica worldwide. The prolonged and frequent use of insecticides produced the selection of insecticide-resistant individuals. The German cockroach is one of the most widespread urban pests in Argentina. In the last decades, resistance monitoring studies in this country demonstrated that there is a high prevalence of pyrethroid-resistant populations of B. germanica in the field. In this work, we studied the resistance mechanisms of a field-collected strain of B. germanica at toxicological, enzymatic, and molecular levels. A resistance ratio of 100 was obtained for the resistant strain when it was exposed to β-cypermethrin. The pretreatment with specific synergists (piperonyl butoxide and triphenyl phosphate) led to a significant increase in the toxicity of the pyrethroid, suggesting an involvement of oxidases and esterases in the detoxification of this insecticide. Moreover, esterase and oxidase activities in the resistant strain were 1.5-fold and 2-fold higher respectively, compared to the susceptible individuals. On the other hand, the voltage-gated sodium channel gene of the resistant cockroaches did not show nucleotidic substitutions in the domain II which are associated to knockdown resistance in this species. These results suggest that the main mechanism of resistance of the studied cockroaches' strain is metabolic, mainly due to an increase in the activity of oxidase and esterase enzymes. The results of this work in addition to other reports found in literature show that the extended use of a single active principle for cockroach control promotes the development of resistance leading to control failure in the field. In contrast, integrated pest management strategies include the use of different control tools in addition to chemical insecticides, which delay the appearance of resistance increasing the efficacy of pest control.

In insects, neuropeptides can act as hormones and neurotransmitters, exerting their physiological roles by activating membrane protein receptors. Because of their relevance in the control of vital processes, the neuroendocrine system of harmful insects could be a potential source of targets for next-generation insecticides, in an integrated and environmentally sustainable management strategy. The study of triatomine neuroendocrinology is of interest to both basic and applied entomology, motivating an active research field. This has been boosted in recent years by the publication of the Rhodnius prolixus genome, which allowed a comprehensive characterization of the neuropeptidergic complement, transcriptomic, proteomic and related molecular and physiological research. Even though research on triatomine species other than R. prolixus is scarce for neuropeptides and their receptors, several publications have emerged in recent years. This allowed performing structural and functional comparisons and proposing both conserved and species-specific characteristics for the subfamily. Here we comprehensively review the triatomine neuropeptidergic system and compare the results obtained for the different species studied to date.

The Indian meal moth Plodia interpunctella is a pest of stored products worldwide. Plant-derived essential oils with insecticidal activity could be safe products to control this species. The scarce information about the mode of action of most plant-derived products limits their use for the control of insect pests. Here, we demonstrate that an essential oil distilled from Lippia turbinata ("poleo") has insecticidal activity on P. interpunctella larvae. Furthermore, we performed a comprehensive characterization of P. interpunctella neuroendocrine system, in comparison with other lepidopteran species.

Background: Rhodnius prolixus has become a model for revealing the molecular bases of insect sensory biology due to the publication of its genome and its well-characterized behavioural repertoire. Gene expression modulation underlies behaviour-triggering processes at peripheral and central levels. Still, the regulation of sensory-related gene transcription in sensory organs is poorly understood. Here we study the genetic bases of plasticity in antennal sensory function, using R. prolixus as an insect model. Results: Antennal expression of neuromodulatory genes such as those coding for neuropeptides, neurohormones and their receptors was characterized in fifth instar larvae and female and male adults by means of RNA-Sequencing (RNASeq). New nuclear receptor and takeout gene sequences were identified for this species, as well as those of enzymes involved in the biosynthesis and processing of neuropeptides and biogenic amines. Conclusions: We report a broad repertoire of neuromodulatory and neuroendocrine-related genes expressed in the antennae of R. prolixus and suggest that they may serve as the local basis for modulation of sensory neuron physiology. Diverse neuropeptide precursor genes showed consistent expression in the antennae of all stages studied. Future studies should characterize the role of these modulatory components acting over antennal sensory processes to assess the relative contribution of peripheral and central regulatory systems on the plastic expression of insect behaviour., Centro Regional de Estudios Genómicos

Vertebrate blood composition is heavily biased towards proteins, and hemoglobin, which is a hemeprotein, is by far the most abundant protein. Typically, hematophagous insects ingest blood volumes several times their weight before the blood meal. This barbarian feast offers an abundance of nutrients, but the degradation of blood proteins generates toxic concentrations of amino acids and heme, along with unparalleled microbiota growth. Despite this challenge, hematophagous arthropods have successfully developed mechanisms that bypass the toxicity of these molecules. While these adaptations allow hematophagous arthropods to tolerate their diet, they also constitute a unique mode of operation for cell signaling, immunity, and metabolism, the study of which may offer insights into the biology of disease vectors and may lead to novel vector-specific control methods.

Cuticle tanning occurs in insects immediately after hatching or molting. During this process, the cuticle becomes dark and rigid due to melanin deposition and protein crosslinking. In insects, different from mammals, melanin is synthesized mainly from dopamine, which is produced from DOPA by the enzyme DOPA decarboxylase. In this work, we report that the silencing of the RpAadc-2 gene, which encodes the putative Rhodnius prolixus DOPA decarboxylase enzyme, resulted in a reduction in nymph survival, with a high percentage of treated insects dying during the ecdysis process or in the expected ecdysis period. Those treated insects that could complete ecdysis presented a decrease in cuticle pigmentation and hardness after molting. In adult females, the knockdown of AADC-2 resulted in a reduction in the hatching of eggs; the nymphs that managed to hatch failed to tan the cuticle and were unable to feed. Despite the failure in cuticle tanning, knockdown of the AADC-2 did not increase the susceptibility to topically applied deltamethrin, a pyrethroid insecticide. Additionally, our results showed that the melanin synthesis pathway did not play a major role in the detoxification of the excess (potentially toxic) tyrosine from the diet, an essential trait for hematophagous arthropod survival after a blood meal., Centro Regional de Estudios Genómicos

The importance of Chagas disease motivated the scientific effort to obtain the complete genomic sequence of the vector species Rhodnius prolixus, this information is also relevant to the understanding of triatomine biology in general. The central nervous system is the key regulator of insect physiology and behavior. Neurohormones (neuropeptides and biogenic amines) are the chemical messengers involved in the regulation and integration of neuroendocrine signals. In insects, this signaling is mainly mediated by the interaction of neurohormone ligands with G protein coupled receptors (GPCRs). The recently sequenced R. prolixus genome provides us with the opportunity to analyze this important family of genes in triatomines, supplying relevant information for further functional studies. Next-generation sequencing methods offer an excellent opportunity for transcriptomic exploration in key organs and tissues in the presence of a reference genome as well as when a reference genome is not available. We undertook a genomic analysis to obtain a genome-wide inventory of opsines and the GPCRs for neurohormones in R. prolixus. Furthermore, we performed a transcriptomic analysis of R. prolixus central nervous system, focusing on neuropeptide precursor genes and neurohormone and opsines GPCRs. In addition, we mined the whole transcriptomes of Triatoma dimidiata, Triatoma infestans and Triatoma pallidipennis - three sanitary relevant triatomine species - to identify neuropeptide precursors and GPCRs genes. Our study reveals a high degree of sequence conservation in the molecular components of the neuroendocrine system of triatomines. Fil: Ons, Sheila. Universidad Nacional de La Plata. Centro Regional de Estudios Genómicos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina Fil: Lavore, Andres Esteban. Universidad Nacional del Noroeste de la Provincia de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Sterkel, Marcos. Universidad Nacional de La Plata. Centro Regional de Estudios Genómicos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidade Federal Do Rio de Janeiro. Instituto de Biología; Brasil Fil: Wulff, Juan Pedro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Centro Regional de Estudios Genómicos; Argentina Fil: Sierra, Ivana Samanta. Universidad Nacional de La Plata. Centro Regional de Estudios Genómicos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina Fil: Martínez Barnetche, Jesús. Centro de Investigaciones Sobre Enfermedades Infecciosa; México Fil: Rodriguez, Mario Henry. Centro de Investigaciones sobre Enfermedades Infecciosas; México Fil: Rivera Pomar, Rolando. Universidad Nacional de La Plata. Centro Regional de Estudios Genómicos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina

Metamorphosis, which depends upon a fine balance between two groups of lipid-soluble hormones such as juvenile hormones (JHs) and ecdysteroids, is an important feature in insect evolution. While it is clear that the onset of metamorphosis depends on the decrease of JH levels, the way in which these hormones exert their activities is not fully understood in Triatominae species. The discovery of a Drosophila melanogaster mutant resistant to the treatment with the JH analogue methoprene, led finally to the description of the methoprene-tolerant gene in Tribolium castaneum (TcMet) as a putative JH receptor. Here we present the genomic and functional characterization of an ortholog of the methoprene-tolerant gene in the hemimetabolous insect Rhodnius prolixus (RpMet). The analysis of the R. prolixus gene showed that the exonic structure is different from that described for holometabolous species, although all the critical protein motifs are well conserved. Expression analysis showed the presence of RpMet mRNA in all the tested tissues: ovary, testis, rectum, Malpighian tubules and salivary glands. When juvenile individuals were treated with RpMet specific double strand RNA (dsRNA), we observed abnormal molting events that resulted in individuals with morphological alterations (adultoids). Similarly, treatment of newly emerged fed females with dsRNA resulted in an abnormal development of the ovaries, with eggs revealing anomalies in size and accumulation of yolk, as well as a decrease in the amount of heme-binding protein. Altogether, our results validate that RpMet is involved in the transduction of JH signaling, controlling metamorphosis and reproduction in R. prolixus. Fil: Villalobos Sambucaro, María José. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Centro Regional de Estudios Genómicos; Argentina. Universidad Nacional de la Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Histología y Embriología Animal; Argentina Fil: Riccillo, Fernando Luis. Universidad Nacional de La Plata. Centro Regional de Estudios Genómicos; Argentina. Universidad Nacional de la Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Histología y Embriología Animal; Argentina Fil: Calderón Fernández, Gustavo Mario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata ; Argentina. Universidad Nacional de la Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Histología y Embriología Animal; Argentina Fil: Sterkel, Marcos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Centro Regional de Estudios Genómicos; Argentina Fil: Diambra, Luis Anibal. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Centro Regional de Estudios Genómicos; Argentina Fil: Ronderos, Jorge Rafael. Universidad Nacional de La Plata. Centro Regional de Estudios Genómicos; Argentina. Universidad Nacional de la Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Histología y Embriología Animal; Argentina

Blood-feeding arthropods are vectors of infectious diseases such as dengue, Zika, Chagas disease, and malaria [1], and vector control is essential to limiting disease spread. Because these arthropods ingest very large amounts of blood, a protein-rich meal, huge amounts of amino acids are produced during digestion. Previous work on Rhodnius prolixus, a vector of Chagas disease, showed that, among all amino acids, only tyrosine degradation enzymes were overexpressed in the midgut compared to other tissues [2]. Here we demonstrate that tyrosine detoxification is an essential trait in the life history of blood-sucking arthropods. We found that silencing Rhodnius tyrosine aminotransferase (TAT) and 4-hydroxyphenylpyruvate dioxygenase (HPPD), the first two enzymes of the phenylalanine/tyrosine degradation pathway, caused the death of insects after a blood meal. This was confirmed by using the HPPD inhibitor mesotrione, which selectively killed hematophagous arthropods but did not affect non-hematophagous insects. In addition, mosquitoes and kissing bugs died after feeding on mice that had previously received a therapeutic effective oral dose (1 mg/kg) of nitisinone, another HPPD inhibitor used in humans for the treatment of tyrosinemia type I [3]. These findings indicate that HPPD (and TAT) can be a target for the selective control of blood-sucking disease vector populations. Because HPPD inhibitors are extensively used as herbicides and in medicine, these compounds may provide an alternative less toxic to humans and more environmentally friendly than the conventional neurotoxic insecticides that are currently used, with the ability to affect only hematophagous arthropods.

We show a straightforward workflow combining homology search in Rhodnius prolixus genome sequence with cloning by rapid amplification of cDNA ends and mass spectrometry. We have identified 32 genes and their transcripts that encode a number of neuropeptide precursors leading to 194 putative peptides. We validated by mass spectrometry 82 of those predicted neuropeptides in the brain of R. prolixus to achieve the first comprehensive genomic, transcriptomic and neuropeptidomic analysis of an insect disease vector. Comparisons of available insect neuropeptide sequences revealed that the R. prolixus genome contains most of the conserved neuropeptides in insects, many of them displaying specific features at the sequence level. Some gene families reported here are identified for the first time in the order Hemiptera, a highly biodiverse group of insects that includes many human, animal and plant disease agents.

Haematophagous insects incorporate a large quantity of blood with each meal, producing a big quantity of urine in a few hours. The activity of the Malpighian tubules (MTs) is facilitated by the increase of the circulation of the haemolymph produced by the increase of the aorta contractions as well as, of the peristaltic waves of the anterior midgut. MTs of Triatoma infestans secrete an allatotropin-like peptide, which has a myostimulatory effect on the hindgut, inducing the mixing and voiding of the content during post-prandial diuresis. We are reporting now the activity of allatotropin (AT) as a cardioacceleratory and a myostimulatory peptide at the level of the anterior midgut. The peptide induced the increase of the rate of contractions of the anterior midgut and the aorta in a wide range of concentrations. The cardioacceleratory effect of AT was dependent on the feeding state of the insects and on the presence of serotonin. The response showed the existence of a differential behavior between sexes, inducing a higher increase on the frequency of contractions, as well as, the width of the aorta in males than in females. Finally, our results suggest that AT interacts with serotonin to facilitate the circulation of haemolymph during post-prandial diuresis.

The voltage-gated sodium channel is the target site of pyrethroid insecticides. Point mutations in the domain II region of the channel have been implicated in pyrethroid resistance of several insect species. We identified the sequence of domain II from the para sodium channel in Rhodnius prolixus , a vector of Chagas’ disease. With this information, we cloned and sequenced the domain II of the sodium channel from the other main Chagas’ disease vector: Triatoma infestans . We also identified the presence of a resistance-conferring mutation (L1014F) in a pyrethroid-resistant population of T. infestans from Argentina, and present a PCR-based method to detect this mutation in individuals from field populations. These findings have important implications for the implementation of strategies for resistance management, and for the rational design of campaigns for the control of Chagas’ disease transmission.