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Background: Aedes aegypti is an urban mosquito vector of dengue and other arboviruses. During epidemic periods, pyrethroid insecticides are used for the control of adult mosquitoes; the worldwide distributed resistance to these insecticides is a cause of failures in vector control campaigns. The primary target of pyrethroids is the voltage-gated sodium channel; point mutations on this channel, called kdr mutations, are associated with pyrethroid resistance. Two kdr mutations, called V1016I and F1534C, augmented in frequency in natural populations of Ae. aegypti from the Americas in the last decade; their association with pyrethroid resistance has been largely demonstrated in field populations throughout the country, and in in vitro assays. The diagnostic of kdr polymorphisms allows an early detection of insecticide resistance spreading, which is critical for timely decisions on vector management. Given the relevance of resistance management, high-throughput methods for kdr genotyping are valuable tools for resistance monitoring programs. These methods should also be cost-effective, to allow regional-scale surveys. Despite the extended presence of Ae. aegypti and the incidence of dengue in Argentina, the presence, abundance and distribution of kdr mutations were not reported in this country up to date. Methods: Aedes aegypti samples were collected from Metropolitan Area of Buenos Aires, and northern localities of Tartagal (Salta province) and Calilegua (Jujuy province). A high resolution melting assay was developed for the simultaneous detection of V1016I and F1534C kdr mutations. Results: We demonstrate the presence of kdrmutations in Argentina in regions under different selection pressure with pyrethroids. We also report a multiplex high-throughput assay based in high resolution melting PCR for the simultaneous genotyping of V1016I and F1534C kdr mutations. Conclusions: We report for the first time the presence of kdr mutations in distant populations from Argentina, with different epidemiological situations and different history of mosquito control efforts. We have developed a high-throughput method for the genotyping of kdr mutations in Ae. aegypti from the American continent. Given its convenient cost and running time, this method can be incorporated in control campaigns for monitoring the presence and spreading of kdr alleles.

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.

BackgroundObligate blood-feeding insects obtain the nutrients and water necessary to ensure survival from the vertebrate blood. The internal taste sensilla, situated in the pharynx, evaluate the suitability of the ingested food. Here, through multiple approaches, we characterized the pharyngeal organ (PO) of the hematophagous kissing bugRhodnius prolixusto determine its role in food assessment. The PO, located antero-dorsally in the pharynx, comprises 8 taste sensilla that become bathed with the incoming blood.ResultsWe showed that these taste sensilla house gustatory receptor neurons projecting their axons through the labral nerves to reach the subesophageal zone in the brain. We found that these neurons are electrically activated by relevant appetitive and aversive gustatory stimuli such as NaCl, ATP and caffeine. Using RNA-Seq, we examined the expression of sensory-related gene families in the PO. We identified gustatory receptors, ionotropic receptors, transient receptor potential channels, pickpocket channels, opsins, takeouts, neuropeptide precursors, neuropeptide receptors and biogenic amine receptors. RNA interference assays demonstrated that the pickpocket channelRproppk014276is necessary for salt detection during feeding.ConclusionWe provide evidence of the role of the pharyngeal organ in food evaluation. This work shows the first comprehensive characterization of a pharyngeal taste organ in a hematophagous insect.

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.

BackgroundAedes aegypti is an urban mosquito vector of Dengue and other arboviruses. During epidemic periods, pyrethroid insecticides are used for the control of adult mosquitoes; the worldwide distributed resistance to these insecticides is a cause of failures in vector control campaigns. The primary target of pyrethroids is the voltage-gated sodium channel; point mutations on this channel, called kdr mutations, are associated with pyrethroid resistance. Two kdr mutations, called V1016I and F1534C, augmented in frequency in natural populations of Ae. aegypti from the Americas in the last decade. The diagnostic of kdr polymorphisms allows an early detection of insecticide resistance spreading, which is critical for timely decisions on vector management. Given the relevance of resistance management, high-throughput methods for kdr genotyping are invaluable tools for resistance monitoring programs. These methods should also be cost-effective, to allow regional-scale surveys. Despite the extended presence of Ae. aegypti and the incidence of dengue in Argentina, the presence, abundance and distribution of kdr mutations were not reported in this country up to date.Methodology and findingsWe report a multiplex high-throughput assay based in High Resolution Melting PCR for the simultaneous genotyping of 1016 and 1534 sites in voltage-gated sodium channel gene. We used this method for the study of individual mosquito samples collected in localities which received different selection pressure with pyrethroids. Compared to other genotyping methods, multiplex High Resolution Melting was high-throughput, cost-efficient, sensitive and specific. We demonstrate for the first time the presence of kdr mutations in Argentina in regions under different selection pressure with pyrethroids.Conclusions and SignificanceWe have developed a high-throughput method for the genotyping of alleles associated with pyrethroid resistance in Ae. aegypti from the American continent. The method developed here is comparable in its sensitivity and reliability with other genotyping methods, but reduces costs and running time. It could be incorporated in control campaigns for control the presence and spreading of resistance-associated alleles. We report here for the first time the presence of kdr mutations in distant populations from Argentina, with different epidemiological situations and different history of mosquito control efforts.Authors summaryAedes aegypti is a mosquito vector of viruses such as dengue, causing millions of infections yearly worldwide. Emergence and distribution of insecticide resistance in this mosquito is a challenge for control campaigns. In this context, the implementation of resistance management strategies became a requisite for successful and sustainable mosquito management. To achieve this objective, it is important to count with early genetic markers of resistance, such as kdr mutations, which are associated with pyrethroid resistance phenotype. Kdr markers can be detected by molecular genotyping diagnostic at early stages in the emergence of resistance; this information should be considered in rational design of control campaigns. Here we present a high-throughput cost-effective method to detect kdr mutations in Ae. aegypti. Also, we demonstrate for the first time the presence of kdr mutations in Argentina.

Background: Hemiptera is one of the most speciose Orders of insects, and the most speciose considering hemimetabolous. 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 plant feeders pentatomids Nezara viridula and Halyomorpha halys; the hematophagous Cimex lectularius, Cimicidae, and Rhodnius prolixus, Reduviidae), and one plant feeder Auchenorryncha (Nilaparvata lugens). Results: Our results point to an expansion of several enzyme families associated to xenobiotic detoxification in heteropterans respect to other species, and present 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 Hemiptera species analyzed here: reduction or even absence of class Epsilon and reduced class Delta in GST superfamily; absence of mitochondrial family CYP12; 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 larger 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 detox-related gene families could shed light on evolutionary forces that shaped their divergence. These families are both important for the detoxification of diet products and to confer tolerance or resistance to synthetic insecticides. Furthermore, we present here the first comprehensive analysis of detoxificant 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 much as for the control of harmful species and the management of insecticide resistance.

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.

BackgroundTriatoma infestans is the main vector of Chagas disease in the Southern Cone. The resistance to pyrethroid insecticides developed by populations of this species impairs the effectiveness of vector control campaigns in wide regions of Argentina. The study of the global transcriptomic response to pyrethroid insecticides is important to deepen the knowledge about detoxification in triatomines.Methodology and findingsWe used RNA-Seq to explore the early transcriptomic response of T. infestans after intoxication with deltamethrin. We were able to assemble a complete transcriptome of this vector and found evidence of differentially expressed genes belonging to diverse families such as chemosensory and odorant-binding proteins, ABC transporters and heat-shock proteins. Moreover, genes related to transcription and translation, energetic metabolism and cuticle rearrangements were also modulated. Finally, we characterized the repertoire of previously uncharacterized detoxification-related gene families in T. infestans and Rhodnius prolixus.Conclusions and significanceOur work contributes to the understanding of the detoxification response in vectors of Chagas disease. Given the absence of genomic information from T. infestans, the analysis presented here constitutes a resource for molecular and physiological studies in this species. The results increase the knowledge on detoxification processes in vectors of Chagas disease, and provide relevant information to explore new potential insecticide resistance mechanisms in these insects.Author summaryChagas disease affects millions of people worldwide. In the Southern Cone, the development of pyrethroid resistant populations from T. infestans is related to vector persistence and affects the efficiency of vector control campaigns. Several studies have explored the causes of insecticide resistance in T. infestans populations. However, the global transcriptomic response after insecticide treatment has not been analyzed in this species so far. In this study, we obtained transcriptomic information which allowed us to characterize important gene families despite the absence of genomic information. Furthermore, we performed a quantitative analysis of gene expression after deltamethrin intoxication. The results provided here increase the knowledge on detoxification processes in vectors of Chagas disease, which is essential for the design of new vector control strategies.

Background: Aedes aegypti (L.) is an urban mosquito, vector of several arboviruses that cause severe diseases in hundreds of million people each year. The resistance to synthetic insecticides developed by Ae. aegypti populations worldwide has contributed to failures in vector control campaigns, increasing the impact of arbovirus diseases. In this context, plant-derived essential oils with larvicidal activity could be an attractive alternative for vector control. However, the mode of action and the detoxificant response of mosquitoes to plant derived compounds have not been established, impairing the optimization of their use. Methods and findings: Here we compare gene expression in Ae. aegypti larvae after 14 hrs of exposure to Eucalyptus camaldulensis essential oil with a control group exposed to vehicle (acetone) for the same lapse, by using RNA-Seq. We found differentially expressed genes encoding for cuticle proteins, fatty-acid synthesis, membrane transporters and detoxificant related gene families (i.e. heat shock proteins, cytochromes P450, glutathione transferases, UDP-glycosyltransferases and ABC transporters). Finally, our RNA-Seq and molecular docking results provide evidence pointing to a central involvement of chemosensory proteins in the detoxificant response in mosquitoes. Conclusions and significance: Our work contributes to the understanding of the physiological response of Ae. aegypti larvae to an intoxication with a natural toxic distilled from Eucalyptus leafs. The results suggest an involvement of most of the gene families associated to detoxification of xenobiotics in insects. Noteworthy, this work provides important information regarding the implication of chemosensory proteins in the detoxification of a natural larvicide. Understanding the mode of detoxification of Eucalyptus distilled compounds could contribute to their implementation as a tool in mosquito control., Los datos de la investigación están disponibles en la base de datos NCBI SRA (número de acceso Bioproject PRJNA671513) (link en "Documentos relacionados"), 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.

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., Centro Regional de Estudios Genómicos

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 southern green stink bug, Nezara viridula is one of the primary soybean pests and causes significant economic losses around the world. In spite of the high proteases inhibitor (PI) levels, N. viridula can feed on developing seeds of field-grown soybean and reduce crop yields. Although the PI-induced responses have been extensively investigated in many pest insects, there is lack of knowledge about the mechanisms that stink bugs employ to withstand cysteine PIs of soybean seeds. This study demonstrated that feeding on developing seeds of field-grown soybean inhibited total proteases activity of N. viridula, as result of inhibition of cathepsin B-like activity in the gut. In addition, from the 30 digestive cathepsins recognized in this study, 6 were identified as cathepsin B-like. Stink bugs that fed on growing seeds of field-grown soybean had similar gut pH to those reared in the laboratory, and both cathepsin B- and L-like had an optima pH of 6.5. Therefore, using specific proteases inhibitors we found that the main proteolytic activity in the gut is from cysteine proteases when N. viridula feeds on soybean crops. Since cathepsin L-like activity was not inhibited by soybean PIs, our results suggested that N. viridula relays on cathepsin L-like to feed on soybean. To our knowledge no study before has shown the impact of seed PIs of field-grown soybean on digestive proteases (cathepsin B- and L-like) of N. viridula. This study suggests that the activity of PI-insensitive cathepsins L-like in the gut would be part of an adaptive strategy to feed on developing soybean seeds. In agreement, the expansions of cathepsin L-like complement observed in pentatomids could confer to the insects a higher versatility to counteract the effects of different PIs.

In animals, neuropeptidergic signaling is essential for the regulation of survival and reproduction. In insects, Orcokinins are poorly studied, despite their high level of conservation among different orders. In particular, there are currently no reports on the role of Orcokinins in the experimental insect model, the fruit fly, Drosophila melanogaster. In the present work, we made use of the genetic tools available in this species to investigate the role of Orcokinins in the regulation of different innate behaviors including ecdysis, sleep, locomotor activity, oviposition, and courtship. We found that RNAi-mediated knockdown of the orcokinin gene caused a disinhibition of male courtship behavior, including the occurrence of male to male courtship, which is rarely seen in wildtype flies. In addition, orcokinin gene silencing caused a reduction in egg production. Orcokinin is emerging as an important neuropeptide family in the regulation of the physiology of insects from different orders. In the case of the fruit fly, our results suggest an important role in reproductive success.

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

The honey bee Apis mellifera is the most abundant managed pollinator in diverse crops worldwide. Consequently, it is exposed to a plethora of environmental stressors, among which are the agrochemicals. In agroecosystems, the herbicide glyphosate (GLY) is one of the most applied. In laboratory assessments, GLY affects the honey bee larval development by delaying its moulting, among other negative effects. However, it is still unknown how GLY affects larval physiology when there are no observable signs of toxicity. We carried out a longitudinal experimental design using the in vitro rearing procedure. Larvae were fed with food containing or not a sub-lethal dose of GLY in chronic exposure (120 h). Individuals without observable signs of toxicity were sampled and their gene expression profile was analyzed with a transcriptomic approach to compare between treatments. Even though 29% of larvae were asymptomatic in the exposed group, they showed transcriptional changes in several genes after the GLY chronic intake. A total of 19 transcripts were found to be differentially expressed in the RNA-Seq experiment, mainly linked with defensive response and intermediary metabolism processes. Furthermore, the enriched functional categories in the transcriptome of the exposed asymptomatic larvae were linked with enzymes with catalytic and redox activity. Our results suggest an enhanced catabolism and oxidative metabolism in honey bee larvae as a consequence of the sub-lethal exposure to GLY, even in the absence of observable symptoms., Centro Regional de Estudios Genómicos

In the kissing bug Rhodnius prolixus, events such as diuresis, antidiuresis, development and reproduction are triggered by blood feeding. Hence, these events can be accurately timed, facilitating physiological experiments. This, combined with its relatively big size, makes R. prolixus an excellent model in insect neuroendocrinological studies. The importance of R. prolixus as a Chagas' disease vector as much as an insect model has motivated the sequencing of its genome in recent years, facilitating genetic and molecular studies. Most crucial physiological processes are regulated by the neuroendocrine system, composed of neuropeptides and their receptors. The identification and characterization of neuropeptides and their receptors could be the first step to find targets for new insecticides. The sequences of 41 neuropeptide precursor genes and the receptors for most of them were identified in the R. prolixus genome. Functional information about many of these molecules was obtained, whereas many neuroendocrine systems are still unstudied in this model species. This review addresses the knowledge available to date regarding the structure, distribution, expression and physiological effects of neuropeptides in R. prolixus, and points to future directions in this research field.

Given that hematophagous insects ingest large quantities of blood in a single meal, they must undergo a rapid post-prandial diuresis in order to maintain homeostasis. In the kissing bug Rhodnius prolixus (Hemiptera: Reduviidae), the coordinated activity of the Malpighian tubules and anterior midgut maintains water and ion balance during the post-prandial diuresis. Three to four hours after the meal the diuretic process finishes, and the animal enters an antidiuretic state to ensure water conservation until the next blood intake. The diuretic and antidiuretic processes are tightly regulated by serotonin and neuropeptides in this insect. In the present work, we report that the neuropeptide precursor CCHamide 2 is involved in the regulation of the post-prandial diuresis in the kissing bug R. prolixus. Our results suggest a dual effect of RhoprCCHamide2 peptide, enhancing the serotonin-induced secretion by Malpighian tubules, and inhibiting serotonin-induced absorption across the anterior midgut. To our knowledge, this is the first report of a hormone presenting opposite effects in the two osmoregulatory organs (i.e. midgut and Malpighian tubules) in insects, probably reflecting the importance of a well-tuned diuretic process in hematophagous insects during different moments after the blood meal., Facultad de Ciencias Exactas, Centro Regional de Estudios Genómicos

Given that hematophagous insects ingest large quantities of blood in a single meal, they must undergo a rapid post-prandial diuresis in order to maintain homeostasis. In the kissing bug

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

Stinkbugs (Hemiptera: Pentatomidae) are of major economic importance as pest of crops. Among the species composing the stinkbug complex, Nezara viridula is one of the most abundant in Brazil, Argentina and the Southern USA. However, this species has been poorly characterized at the genetic and physiological level. Here we sequenced and analyzed the complete transcriptome of N. viridula male and female adults. We identifed neuropeptide precursor genes and G-protein coupled receptors for neuropeptides in this transcriptome. Mature neuropeptides were identifed in N. viridula brain extracts by liquid chromatography-tandem mass spectrometry. We also analyzed the neuropeptide precursor complement in the genome sequence of Halyomorpha halys, another pentatomid of economic relevance. We compared the results in both pentatomids with the well-characterized neuropeptide repertoire from the kissing bug Rhodnius prolixus (Hemiptera: Reduviidae). We identifed both groupspecifc features (which could be related to the diferent feeding habits) and similarities that could be characteristic of Heteroptera. This work contributes to a deeper knowledge of the genetic information of these pests, with a focus on neuroendocrine system characterization., 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

Ecdysis is a vital process for insects, during which they shed the old cuticle in order to emerge as the following developmental stage. Given its relevance for survival and reproduction, ecdysis is tightly regulated by peptidic hormones that conform an interrelated neuromodulatory network. This network was studied in species that undergo a complete metamorphosis, but not in hemimetabola. In a recent work, we demonstrated that orcokinin neuropeptides are essential for ecdysis to occur in the kissing bug Rhodnius prolixus. Here we performed gene silencing, quantitative PCR and in vitro treatments in order to study the interrelationships between RhoprOKs and hormones such as ecdysis triggering hormone, corazonin, eclosion hormone, crustacean cardioactive peptide and ecdysone. Our results suggest that RhoprOKs directly or indirectly regulate the expression of other genes. Whereas RhoprOKA is centrally involved in the regulation of gene expression, RhoprOKB is implicated in processes related to midgut physiology. Therefore, we propose that the different transcripts encoded in RhoprOK gene could integrate signaling cues, in order to coordinate the nutritional state with development and ecdysis. Given the emerging data that point to OKs as important factors for survival and reproduction, they could be candidates in the search for new insect management strategies based on neuroendocrine targets. Fil: Wulff, Juan Pedro. Universidad Nacional de La Plata. Centro Regional de Estudios Genómicos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Capriotti, Natalia. Universidad Nacional de La Plata. Centro Regional de Estudios Genómicos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Ons, Sheila. Universidad Nacional de La Plata. Centro Regional de Estudios Genómicos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

To grow and develop insects must undergo ecdysis. During this process, the individual sheds the old cuticle to emerge as the following developmental stage. During ecdysis, different programed behaviors are regulated by neuropeptidergic pathways. In general, components of these pathways are better characterized in crustacean and holometabolous insects than in hemimetabola. In insects, the orkoninin gene produces two different neuropeptide precursors by alternative splicing: orcokinin A and orcokinin B. Although orcokinins are well conserved in insect species, their physiological role remains elusive. Here we describe a new splicing variant of the orcokinin gene in the hemimetabolous triatomine Rhodnius prolixus. We further analyze the expression pattern and the function of the alternatively spliced RhoprOK transcripts by means of immunohistochemistry and RNAi-mediated gene silencing. Our results indicate that orkoninis play an essential role in the peptidergic signaling pathway regulating ecdysis in the hemimetabolous insect Rhodnius prolixus., Centro Regional de Estudios Genómicos, Centro de Endocrinología Experimental y Aplicada

Point mutations in the voltage-gated sodium channel, the primary target of pyrethroid insecticides, have been associated with the resistance in Triatoma infestans, an important vector of Chagas' disease. Hence, the sustainability of vector control programs requires the implementation of resistance management strategies.We determined the sensitivity of the molecular assays previously designed for early resistance detection to be used in pooled samples from a wide area of the endemic region, and validated them for their routine use in control campaigns for the monitoring of insecticide resistance in T. infestans. Consequently, we used these methods to examine the distribution of resistance-associated mutations in the sodium channel gene in populations of T. infestans from the Argentinean and Bolivian Gran Chaco.The PASA and REA assays tested proved sensitive enough to detect kdr SNPs in pooled samples, indicating these assays are suitable for routine screening in insecticide resistance surveillance. Two geographically differentiated foci were detected in T. infestans populations from the Argentinean and Bolivian Gran Chaco, with populations on the Bolivian-Argentinean border carrying L1014F mutation, and those from the Argentinean Chaco carrying L925I mutation. In all highly resistant populations analyzed, one of both kdr mutations was present, and toxicological assays determined that all pyrethroid resistant populations analyzed herein were sensitive to fenitrothion.The principal cause of pyrethroid resistance in T. infestans from the Gran Chaco ecoregion is kdr mutations in the sodium channel. Different levels of resistance occur in different populations carrying identical mutation, suggesting the existence of contributory mechanisms. Fil: Sierra, Ivana Samanta. 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: Capriotti, Natalia. 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: Fronza, Georgina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones Científicas y Técnicas para la Defensa. Centro de Investigación de Plagas e Insecticidas; Argentina. Ministerio de Defensa. Instituto de Investigaciones Científicas y Técnicas para la Defensa; Argentina Fil: Mougabure Cueto, Gastón Adolfo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones Científicas y Técnicas para la Defensa. Centro de Investigación de Plagas e Insecticidas; Argentina. Ministerio de Defensa. Instituto de Investigaciones Científicas y Técnicas para la Defensa; Argentina Fil: Ons, Sheila. 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

Peptides may act through G protein-coupled receptors to influence cardiovascular performance; thus, delineating mechanisms involved in signaling is a molecular-based strategy to influence health. Molecular switches, often represented by conserved motifs, maintain a receptor in an inactive state. However, once the switch is broken, the transmembrane regions move and activation occurs. The molecular switches of Drosophila melanogaster myosuppressin (MS) receptors were previously identified to include a unique ionic lock and novel 3-6 lock, as well as transmission and tyrosine toggle switches. In addition to MS, cardioactive ligands structurally related by a C-terminal RF-NH2 include sulfakinin, neuropeptide F (NPF), short NPF, and FMRF-NH2-containing peptide subfamilies. We hypothesized receptor molecular switch motifs were conserved within a RF-NH2 subfamily and across species. Thus, we investigated RF-NH2 receptor (RFa-R) molecular switches in D. melanogaster, Tribolium castaneum, Anopheles gambiae, Rhodnius prolixus, and Bombyx mori. Adipokinetic hormone (AKH), which does not contain a RF-NH2, was also examined. The tyrosine toggle switch and ionic lock showed a higher degree of conservation within a RF-NH2 subfamily than the transmission switch and 3-7 lock. AKH receptor motifs were not representative of a RF-NH2 subfamily. The motifs and interactions of switches in the RFa-Rs were consistent with receptor activation and ligand-specific binding.

Orcokinins (OKs) are neuropeptides that were first identified in crustacean through their myotropic activity. In insects, the OK gene gives rise to two mRNAs coding for two different families of conserved mature neuropeptides: OKA and OKB. Although OKs are conserved in many insect species, its physiological role in this animal class is not fully understood. Until now prothoracicotropic, regulatory of light entrainment to the circadian clock and >awakening> activities have been reported for these peptides in different insect species. Here we report the identification of OKA and OKB precursors in the cockroach Blattella germanica. OKA mRNA was detected in brain, whereas OKB mRNA was detected both in brain and midgut. In vivo silencing of OK precursors suggests the involvement of OK gene products in the regulation of vitellogenin expression in the fat body, an action that appears to be independent of juvenile hormone. This is the first time that a function of this kind has been reported for OKs., This work was supported by the Spanish MICINN (Grants CGL2008-03517/BOS to XB and BFU2010-15906 to JLM) and MINECO (Grant CGL2012-36251 to XB), and Catalan Government (2014 SGR 619). The research has also benefited from FEDER funds. SO was recipient of an external fellowship from National Council of Scientific and Technical Research (CONICET-Argentina) to work in the Institute of Evolutionary Biology, in Barcelona.

Peptidergic signaling regulates cardiac contractility; thus, identifying molecular switches, ligand-receptor contacts, and antagonists aids in exploring the underlying mechanisms to influence health. Myosuppressin (MS), a decapeptide, diminishes cardiac contractility and gut motility. Myosuppressin binds to G protein-coupled receptor (GPCR) proteins. Two Drosophila melanogaster myosuppressin receptors (DrmMS-Rs) exist; however, no mechanism underlying MS-R activation is reported. We predicted DrmMS-Rs contained molecular switches that resembled those of Rhodopsin. Additionally, we believed DrmMS-DrmMS-R1 and DrmMS-DrmMS-R2 interactions would reflect our structure-activity relationship (SAR) data. We hypothesized agonist- and antagonist-receptor contacts would differ from one another depending on activity. Lastly, we expected our study to apply to other species; we tested this hypothesis in Rhodnius prolixus, the Chagas disease vector. Searching DrmMS-Rs for molecular switches led to the discovery of a unique ionic lock and a novel 3-6 lock, as well as transmission and tyrosine toggle switches. The DrmMS-DrmMS-R1 and DrmMS-DrmMS-R2 contacts suggested tissue-specific signaling existed, which was in line with our SAR data. We identified R. prolixus (Rhp)MS-R and discovered it, too, contained the unique myosuppressin ionic lock and novel 3-6 lock found in DrmMS-Rs as well as transmission and tyrosine toggle switches. Further, these motifs were present in red flour beetle, common water flea, honey bee, domestic silkworm, and termite MS-Rs. RhpMS and DrmMS decreased R. prolixus cardiac contractility dose dependently with EC50 values of 140 nM and 50 nM. Based on ligand-receptor contacts, we designed RhpMS analogs believed to be an active core and antagonist; testing on heart confirmed these predictions. The active core docking mimicked RhpMS, however, the antagonist did not. Together, these data were consistent with the unique ionic lock, novel 3-6 lock, transmission switch, and tyrosine toggle switch being involved in mechanisms underlying TM movement and MS-R activation, and the ability of MS agonists and antagonists to influence physiology., Facultad de Ciencias Exactas

Rhodnius prolixus not only has served as a model organism for the study of insect physiology, but also is a major vector of Chagas disease, an illness that affects approximately seven million people worldwide. We sequenced the genome of R. prolixus, generated assembled sequences covering 95% of the genome (∼702 Mb), including 15,456 putative protein-coding genes, and completed comprehensive genomic analyses of this obligate blood-feeding insect. Although immunedeficiency (IMD)-mediated immune responses were observed, R. prolixus putatively lacks key components of the IMD pathway, suggesting a reorganization of the canonical immune signaling network. Although both Toll and IMD effectors controlled intestinal microbiota, neither affected Trypanosoma cruzi, the causal agent of Chagas disease, implying the existence of evasion or tolerance mechanisms. R. prolixus has experienced an extensive loss of selenoprotein genes, with its repertoire reduced to only two proteins, one of which is a selenocysteine-based glutathione peroxidase, the first found in insects. The genome contained actively transcribed, horizontally transferred genes from Wolbachia sp., which showed evidence of codon use evolution toward the insect use pattern. Comparative protein analyses revealed many lineage-specific expansions and putative gene absences in R. prolixus, including tandem expansions of genes related to chemoreception, feeding, and digestion that possibly contributed to the evolution of a blood-feeding lifestyle. The genome assembly and these associated analyses provide critical information on the physiology and evolution of this important vector species and should be instrumental for the development of innovative disease control methods., La lista completa de autores que integran el documento puede consultarse en el archivo, Este documento tiene una corrección (ver documento relacionado)., Centro Regional de Estudios Genómicos, Instituto de Investigaciones Bioquímicas de La Plata

Background: Triatomine insects are vectors of Trypanosoma cruzi, a protozoan parasite that is the causative agent of Chagas’ disease. This is a neglected disease affecting approximately 8 million people in Latin America. The existence of diverse pyrethroid resistant populations of at least two species demonstrates the potential of triatomines to develop high levels of insecticide resistance. Therefore, the incorporation of strategies for resistance management is a main concern for vector control programs. Three enzymatic superfamilies are thought to mediate xenobiotic detoxification and resistance: Glutathione Transferases (GSTs), Cytochromes P450 (CYPs) and Carboxyl/Cholinesterases (CCEs). Improving our knowledge of key triatomine detoxification enzymes will strengthen our understanding of insecticide resistance processes in vectors of Chagas’ disease. Methods and findings: The discovery and description of detoxification gene superfamilies in normalized transcriptomes of three triatomine species: Triatoma dimidiata, Triatoma infestans and Triatoma pallidipennis is presented. Furthermore, a comparative analysis of these superfamilies among the triatomine transcriptomes and the genome of Rhodnius prolixus, also a triatomine vector of Chagas’ disease, and other well-studied insect genomes was performed. The expression pattern of detoxification genes in R. prolixus transcriptomes from key organs was analyzed. The comparisons reveal gene expansions in Sigma class GSTs, CYP3 in CYP superfamily and clade E in CCE superfamily. Moreover, several CYP families identified in these triatomines have not yet been described in other insects. Conversely, several groups of insecticide resistance related enzymes within each enzyme superfamily are reduced or lacking in triatomines. Furthermore, our qRT-PCR results showed an increase in the expression of a CYP4 gene in a T. infestans population resistant to pyrethroids. These results could point to an involvement of metabolic detoxification mechanisms on the high levels of pyrethroid resistance detected in triatomines from the Gran Chaco ecoregion. Conclusions and significance: Our results help to elucidate the potential insecticide resistance mechanisms in vectors of Chagas’ disease and provide new relevant information for this field. This study shows that metabolic resistance might be a contributing cause of the high pyrethroid resistance observed in wild T. infestans populations from the Gran Chaco ecoregion, area in which although subjected to intense pyrethroid treatments, vector control has failed. This study opens new avenues for further functional studies on triatomine detoxification mechanisms., Centro Regional de Estudios Genómicos, Centro de Endocrinología Experimental y Aplicada