The pharyngeal taste organ of a blood-feeding insect functions in food recognition.

BACKGROUND: Obligate 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 bug Rhodnius prolixus to determine its role in food assessment. The PO, located antero-dorsally in the pharynx, comprises eight taste sensilla that become bathed with the incoming blood. RESULTS: We 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 salt-related pickpocket channel Rproppk014276 is required during feeding of an appetitive solution of NaCl and ATP. CONCLUSIONS: We provide evidence of the role of the pharyngeal organ in food evaluation. This work shows a comprehensive characterization of a pharyngeal taste organ in a hematophagous insect.

Silica-coated magnetic particles for efficient RNA extraction for SARS-CoV-2 detection.

Molecular diagnostic methods to detect and quantify viral RNA in clinical samples rely on the purification of the genetic material prior to reverse transcription polymerase chain reaction (qRT-PCR). Due to the large number of samples processed in clinical laboratories, automation has become a necessity in order to increase method processivity and maximize throughput per unit of time. An attractive option for isolating viral RNA is based on the magnetic solid phase separation procedure (MSPS) using magnetic microparticles. This method offers the advantage over other alternative methods of making it possible to automate the process. In this study, we report the results of the MSPS method based on magnetic microparticles obtained by a simple synthesis process, to purify RNA from oro- and nasopharyngeal swab samples of patients suspected of COVID-19 provided by three diagnostic laboratories located in the Buenos Aires Province, Argentina. Magnetite nanoparticles of Fe3O4 (MNPs) were synthesized by the coprecipitation method and then coated with silica (SiO2) produced by hydrolysis of tetraethyl orthosilicate (TEOS). After preliminary tests on samples from the A549 human lung cell line and swabs, an extraction protocol was developed. The quantity and purity of the RNA obtained were determined by gel electrophoresis, spectrophotometry, and qRT-PCR. Tests on samples from naso- and oropharyngeal swabs were performed in order to validate the method for RNA purification in high-throughput SARS-CoV-2 diagnosis by qRT-PCR. The method was compared to the spin columns method and the automated method using commercial magnetic particles. The results show that the method developed is efficient for RNA extraction from nasal and oropharyngeal swab samples, and also comparable to other extraction methods in terms of sensitivity for SARS-CoV-2 detection. Of note, this procedure and reagents developed locally were intended to overcome the shortage of imported diagnostic supplies as the sudden spread of COVID-19 required unexpected quantities of nucleic acid isolation and diagnostic kits worldwide.

First report on knockdown resistance mutations in wild populations of Aedes aegypti from Argentina determined by a novel multiplex high-resolution melting polymerase chain reaction method.

BACKGROUND: The mosquito Aedes aegypti is an urban vector of dengue and other arboviruses. During epidemics of these viruses, pyrethroid insecticides are used for the control of adult mosquitoes. The worldwide resistance of Ae. aegypti to these insecticides is a cause of failure of vector control campaigns. The primary target of pyrethroids is the voltage-gated sodium channel. Point mutations in the gene coding for this channel, called knockdown resistance (kdr) mutations, are associated with pyrethroid resistance. Two kdr mutations, V1016I and F1534C, have increased in frequency in natural populations of Ae. aegypti in the Americas during the last decade. Their association with pyrethroid resistance has been largely demonstrated in field populations throughout the Americas, and in in vitro assays. Diagnostics for kdr polymorphism allow early detection of the spread of insecticide resistance, which is critical for timely decisions on vector management. Given the importance of resistance management, high-throughput methods for kdr genotyping are valuable tools as they can be used for resistance monitoring programs. These methods should be cost-effective, to allow regional-scale surveys. Despite the extensive presence of Ae. aegypti and incidence of dengue in Argentina, the presence, abundance, and distribution of kdr mutations in populations of this mosquito have yet to be reported for the country. METHODS: Aedes aegypti samples were collected as immature stages or adults from Buenos Aires Metropolitan Area and northern localities of Tartagal (Salta Province) and Calilegua (Jujuy Province). Immature stages were maintained in the laboratory until they developed into adults. A high-resolution melting assay, based on an analysis of melting temperatures, was developed for the simultaneous genotyping of V1016I and F1534C kdr mutations. We used this method to infer the presence and frequencies of kdr alleles in 11 wild populations from Argentina. RESULTS: We demonstrated the presence of kdr mutations in Ae. aegypti in Argentina in regions where this species is under different selection pressures due to the use of pyrethroids. The populations under analysis are located in geographically distant regions of the species' distribution in Argentina: the northern provinces of Salta and Jujuy and the Buenos Aires Metropolitan Area. Higher frequencies of resistant-associated alleles were detected in the northern region. We report a multiplex high-throughput assay based on a high-resolution melting polymerase chain reaction method for the simultaneous genotyping of V1016I and F1534C kdr mutations. This assay was shown to be cost-effective, and thus provides an interesting molecular tool for kdr genotyping in A. aegypti control campaigns. CONCLUSIONS: We report, to the best of our knowledge for the first time, the presence of kdr mutations in populations of Ae. aegypti from geographically distant locations of Argentina that differ with respect to their epidemiological situation and history of mosquito control. We have developed a high-throughput method for the genotyping of kdr mutations in Ae. aegypti from the Americas. Given its affordability and short running time, this method can be used in control campaigns to monitor the presence and spread of kdr alleles. The information provided here is relevant for the rational design of control strategies in the context of integrated vector management.

eIF3 subunit M regulates blood meal digestion in Rhodnius prolixus affecting ecdysis, reproduction, and survival.

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 2 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 molting 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.

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

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

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

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

Transcriptomic modulation in response to an intoxication with deltamethrin in a population of Triatoma infestans with low resistance to pyrethroids.

BACKGROUND: Triatoma 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 FINDINGS: We used RNA-Seq to explore the early transcriptomic response after intoxication with deltamethrin in a population of T. infestans which presents low resistance to pyrethroids. 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 SIGNIFICANCE: Our work contributes to the understanding of the detoxification response in vectors of Chagas disease. Given the absence of an annotated genome 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 undescribed potential insecticide resistance mechanisms in populations of these insects.

Characterization of the pyrethroid resistance mechanisms in a Blattella germanica (Dictyoptera: Blattellidae) strain from Buenos Aires (Argentina).

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 ß-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.

Orcokinin neuropeptides regulate reproduction in the fruit fly, Drosophila melanogaster.

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.

Transcriptomic analysis and molecular docking reveal genes involved in the response of Aedes aegypti larvae to an essential oil extracted from Eucalyptus.

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.

Proteases inhibitors-insensitive cysteine proteases allow Nezara viridula to feed on growing seeds of field-grown soybean.

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.

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.

Plodia interpunctella (Lepidoptera: Pyralidae): Intoxication with essential oils isolated from Lippia turbinata (Griseb.) and analysis of neuropeptides and neuropeptide receptors, putative targets for pest control.

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.

Chronic exposure to glyphosate induces transcriptional changes in honey bee larva: A toxicogenomic study.

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.

Transcriptomics supports local sensory regulation in the antenna of the kissing-bug Rhodnius prolixus.

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 (RNA-Seq). 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.

The neuropeptide CCHamide2 regulates diuresis in the Chagas disease vector Rhodnius prolixus.

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 CCHamide2 is involved in the regulation of the post-prandial diuresis in 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.

DOPA decarboxylase is essential for cuticle tanning in Rhodnius prolixus (Hemiptera: Reduviidae), affecting ecdysis, survival and reproduction.

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.

Nezara viridula (Hemiptera: Pentatomidae) transcriptomic analysis and neuropeptidomics.

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 identified neuropeptide precursor genes and G-protein coupled receptors for neuropeptides in this transcriptome. Mature neuropeptides were identified 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 identified both group-specific features (which could be related to the different 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.

Orcokinins regulate the expression of neuropeptide precursor genes related to ecdysis in the hemimetabolous insect Rhodnius prolixus.

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.