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

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

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

Sugar feeding in triatomines: a new perspective for controlling the transmission of Chagas disease.

Introduction: Triatomines are vectors of Trypanosoma cruzi, the etiological agent of Chagas disease. Currently, there is no vaccine against this disease. Thus, control of the insect vector population is the main strategy available to reduce the number of cases. Triatomines are considered obligate hematophagous, but different alternative feeding behaviors were described, such as haemolymphagy or plant feeding. Methods: To determine the preference for sugar feeding in nymphs and adults of Rhodnius prolixus, the insects were exposed a piece of cotton containing bromophenol blue plus sucrose. In addition, we offered several sugars for different species of triatomines, and tested sugar meals as a route of delivery of insecticides in first-instar nymphs of R. prolixus. The effect of sugar feeding on the physiology of these different species of triatomines was recorded. Results: First instar nymphs ingested sucrose more strongly than other stages, and showed high mortality rates. In different species of triatomines, sucrose induced an ingestion, but engorgement varied according to the species. R. prolixus nymphs showed an indiscriminate intake of various sugars, with very different physiological effects. Furthermore, ingesting different combinations of insecticides + sugar significantly reduced insect survival. Discussion: In summary, we described for the first-time sugar feeding as a widespread behavior in several species of triatomines, and the possibility of the use of toxic sugar baits for the control of these vectors. The knowledge of feeding behavior in these insects can be fundamental for the development of new strategies to control Chagas disease.

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

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

Trypanosoma cruzi infection modulates secreted phospholipase A2 expression in the salivary glands of Rhodnius prolixus.

Phospholipases A2 (PLA2) comprise a superfamily of enzymes that specifically catalyze hydrolysis of the ester bond at the sn-2 position of glycerophospholipids, generating lysophospholipids and fatty acids. In Rhodnius prolixus, one of the main vectors of the Chagas's disease etiologic agent Trypanosoma cruzi, it was previously shown that lysophosphatidylcholine, a bioactive lipid, found in the insect's saliva, contributes to the inhibition of platelet aggregation, and increases the production of nitric oxide, an important vasodilator. Due to its role in potentially generating LPC, here we studied the PLA2 present in the salivary glands of R. prolixus. PLA2 activity is approximately 100 times greater in the epithelium than in the contents of salivary glands. Our study reveals the role of the RpPLA2XIIA gene in the insect feeding performance and in the fatty acids composition of phospholipids extracted from the salivary glands. Knockdown of RpPLA2XIIA significantly altered the relative amounts of palmitic, palmitoleic, oleic and linoleic acids. A short-term decrease in the expression of RpPLA2III and RpPLA2XIIA in the salivary glands of R. prolixus was evident on the third day after infection by T. cruzi. Taken together, our results contribute to the understanding of the role of PLA2 in the salivary glands of hematophagous insects and show that the parasite is capable of modulating even tissues that are not colonized by it.

Export of heme by the feline leukemia virus C receptor regulates mitochondrial biogenesis and redox balance in the hematophagous insect Rhodnius prolixus.

Heme is a prosthetic group of proteins involved in vital physiological processes. It participates, for example, in redox reactions crucial for cell metabolism due to the variable oxidation state of its central iron atom. However, excessive heme can be cytotoxic due to its prooxidant properties. Therefore, the control of intracellular heme levels ensures the survival of organisms, especially those that deal with high concentrations of heme during their lives, such as hematophagous insects. The export of heme initially attributed to the feline leukemia virus C receptor (FLVCR) has recently been called into question, following the discovery of choline uptake by the same receptor in mammals. Here, we found that RpFLVCR is a heme exporter in the midgut of the hematophagous insect Rhodnius prolixus, a vector for Chagas disease. Silencing RpFLVCR decreased hemolymphatic heme levels and increased the levels of intracellular dicysteinyl-biliverdin, indicating heme retention inside midgut cells. FLVCR silencing led to increased expression of heme oxygenase (HO), ferritin, and mitoferrin mRNAs while downregulating the iron importers Malvolio 1 and 2. In contrast, HO gene silencing increased FLVCR and Malvolio expression and downregulated ferritin, revealing crosstalk between heme degradation/export and iron transport/storage pathways. Furthermore, RpFLVCR silencing strongly increased oxidant production and lipid peroxidation, reduced cytochrome c oxidase activity, and activated mitochondrial biogenesis, effects not observed in RpHO-silenced insects. These data support FLVCR function as a heme exporter, playing a pivotal role in heme/iron metabolism and maintenance of redox balance, especially in an organism adapted to face extremely high concentrations of heme.

In the fed state, autophagy plays a crucial role in assisting the insect vector Rhodnius prolixus mobilize TAG reserves under forced flight activity.

Autophagy is a cellular degradation pathway mediated by highly conserved autophagy-related genes (Atgs). In our previous work, we showed that inhibiting autophagy under starvation conditions leads to significant physiological changes in the insect vector of Chagas disease Rhodnius prolixus; these changes include triacylglycerol (TAG) retention in the fat body, reduced survival and impaired locomotion and flight capabilities. Herein, because it is known that autophagy can be modulated in response to various stimuli, we further investigated the role of autophagy in the fed state, following blood feeding. Interestingly, the primary indicator for the presence of autophagosomes, the lipidated form of Atg8 (Atg8-II), displayed 20%-50% higher autophagic activation in the first 2 weeks after feeding compared to the third week when digestion was complete. Despite the elevated detection of autophagosomes, RNAi-mediated suppression of RpAtg6 and RpAtg8 did not cause substantial changes in TAG or protein levels in the fat body or the flight muscle during blood digestion. We also found that knockdown of RpAtg6 and RpAtg8 led to modest modulations in the gene expression of essential enzymes involved in lipid metabolism and did not significantly stimulate the expression of the chaperones BiP and PDI, which are the main effectors of the unfolded protein response. These findings indicate that impaired autophagy leads to slight disturbances in lipid metabolism and general cell proteostasis. However, the ability of insects to fly during forced flight until exhaustion was reduced by 60% after knockdown of RpAtg6 and RpAtg8. This change was accompanied by TAG and protein increases as well as decreased ATP levels in the fat body and flight muscle, indicating that autophagy during digestion, i.e., under fed conditions, is necessary to sustain high-performance activity.

Local age-dependent neuromodulation in Rhodnius prolixus antennae.

Kissing bugs do not respond to host cues when recently molted and only exhibit robust host-seeking several days after ecdysis. Behavioral plasticity has peripheral correlates in antennal gene expression changes through the week after ecdysis. The mechanisms regulating these peripheral changes are still unknown, but neuropeptide, G-protein coupled receptor, nuclear receptor, and takeout genes likely modulate peripheral sensory physiology. We evaluated their expression in antennal transcriptomes along the first week postecdysis of Rhodnius prolixus 5th instar larvae. Besides, we performed clustering and co-expression analyses to reveal relationships between neuromodulatory (NM) and sensory genes. Significant changes in transcript abundance were detected for 50 NM genes. We identified 73 sensory-related and NM genes that were assigned to nine clusters. According to their expression patterns, clusters were classified into four groups: two including genes up or downregulated immediately after ecdysis; and two with genes with expression altered at day 2. Several NM genes together with sensory genes belong to the first group, suggesting functional interactions. Co-expression network analysis revealed a set of genes that seem to connect with sensory system maturation. Significant expression changes in NM components were described in the antennae of R. prolixus after ecdysis, suggesting that a local NM system acts on antennal physiology. These changes may modify the sensitivity of kissing bugs to host cues during this maturation interval.

Deficiency of Brummer lipase disturbs lipid mobilization and locomotion, and impairs reproduction due to defects in the eggshell ultrastructure in the insect vector Rhodnius prolixus.

Rhodnius prolixus is a hematophagous insect, which feeds on large and infrequent blood meals, and is a vector of trypanosomatids that cause Chagas disease. After feeding, lipids derived from blood meal are stored in the fat body as triacylglycerol, which is recruited under conditions of energy demand by lipolysis, where the first step is catalyzed by the Brummer lipase (Bmm), whose orthologue in mammals is the adipose triglyceride lipase (ATGL). Here, we investigated the roles of Bmm in adult Rhodnius prolixus under starvation, and after feeding. Its gene (RhoprBmm) was expressed in all the analyzed insect organs, and its transcript levels in the fat body were not altered by nutritional status. RNAi-mediated knockdown of RhoprBmm caused triacylglycerol retention in the fat body during starvation, resulting in larger lipid droplets and lower ATP levels compared to control females. The silenced females showed decreased flight capacity and locomotor activity. When RhoprBmm knockdown occurred before the blood meal and the insects were fed, the females laid fewer eggs, which collapsed and showed low hatching rates. Their hemolymph had reduced diacylglycerol content and vitellogenin concentration. The chorion (eggshell) of their eggs had no difference in hydrocarbon amounts or in dityrosine crosslinking levels compared to control eggs. However, it showed ultrastructural defects. These results demonstrated that Bmm activity is important not only to guarantee lipid mobilization to maintain energy homeostasis during starvation, but also for the production of viable eggs after a blood meal, by somehow contributing to the right formation of the egg chorion.

Lipid metabolism dynamic in Triatomine Rhodnius prolixus during acute Trypanosoma rangeli infection.

During its life cycle, Trypanosoma rangeli invades the hemolymph of its invertebrate host and colonizes hemocytes and salivary glands. The parasite cannot synthesize some lipid classes, and during its cycle, it depends on the uptake of these molecules from its vertebrate and invertebrate hosts to meet growth and differentiation requirements. However, until now, knowledge on how the parasite affects the lipid physiology of individual insect organs has been largely unknown. Herein, the biochemical and molecular dynamics of triatomine R. prolixus lipid metabolism in response to acute T. rangeli infection were investigated. Biochemical and microscopic assays revealed the lipid droplet profile and the levels of the different identified lipid classes. In addition, a qRT‒PCR approach was used to determine the expression profile of 6 protein-coding genes involved in the R. prolixus lipid physiology. We observed that triacylglycerol (TAG), monoacylglycerol (MAG), phosphatidylethanolamine (PE) and phosphatidylcholine (PC) levels in the fat body decreased in infected insects. On the other hand, high levels of free fatty acids were observed in the hemolymph during infection. Analysis by confocal microscopy revealed a decrease in lipid droplets size from infected fat bodies, and investigations by scanning electron microscopy revealed a significant number of parasites adhered to the surface of the organ. T. rangeli infection upregulated the transcript levels of the protein-coding gene for the acetyl-CoA carboxylase, the first enzyme in the de novo fatty acid synthesis pathway, responsible for the production of malonyl-CoA. On the other hand, downregulation of lipophorin receptor was observed. In conclusion, this study reveals a new set of molecular events that occur within the vector in response to the challenge imposed by the parasite.

Allatotropic peptides modulate muscle contraction of the female reproductive system in Rhodnius prolixus (STÄL).

Allatotropin (AT) acts as a myoregulator at the level of the dorsal vessel (DV) and midgut (MG) in triatominae insects. Previous analyses of the expression of the AT receptor in Rhodnius prolixus showed that AT is expressed in the DV and MG, but also in the reproductive system in females. To further study the activity of AT on female reproductive organs we analyzed the response by adult females in different physiological conditions, including unfed (virgin and mated), and fed mated females (gravid), to doses ranging between 10-14 and 10-6M. Myoregulatory activity was evaluated in vivo, by recording independently the frequency of contractions of each organ after treatment. The results show that the effect of AT varies depending on the organs and on the physiological state of the female. Whilst unfed virgin females did not show response to the peptide for neither of the applied doses, the ovaries showed a differential response, presenting the highest frequency of contractions in gravid individuals. An increase in the frequency of contractions of the oviducts was only observed in mated females. Uterus and spermathecae responded in both gravid and mated females, with maximum activity in the latter. In the bursa, responses were only detected in gravid females. The differential response of the organs seems to be associated to particular moments along the reproductive cycle, such as with the spermathecae that reacted to AT in both unfed mated and gravid females, when the movement of spermatozoids is physiologically crucial. Testes and accessory glands of the male, expressed the mRNA of AT precursor, suggesting that the male would modulate the contractile behavior of the female reproductive system after copula. The ovaries also expressed AT mRNA suggesting the existence of a paracrine/autocrine system modulating muscle contraction.

Digestive α-L-fucosidase activity in Rhodnius prolixus after blood feeding: effect of secretagogue and nutritional stimuli.

Introduction: Rhodnius prolixus (Hemiptera: Reduviidae) is an important vector of Trypanosoma cruzi, the causative agent of Chagas Disease. This insect is a model for the study of insect physiology, especially concerning the digestion of blood. Among the enzymes produced in the midgut of R. prolixus after blood feeding there is a α-L-fucosidase activity. There are very few studies on α-L-fucosidase of insects, and the role of R. prolixus α-L-fucosidase is still not clear. In this work, we tested if the mechanism for production of this enzyme is similar to the observed for proteases, a secretatogue mechanism that respond to the protein contents of the meal. Methods: We tested if specific proteins or sugars elicit this response, which may help to understand the nature of the physiological substrate for this enzyme. Results: In general, our results showed that the Anterior Midgut was the only midgut fraction that responds to the blood meal in terms of α-L-fucosidase production. Besides that, this response was not triggered by midgut distension or by ingestion of the blood cell fraction. Conversely, the enzyme was produced after feeding with the plasma fraction. However, the production of α-L-fucosidase was also triggered by different biochemical stimuli, as protein or fucoidan ingestion. Discussion: This suggested that the production of the enzyme in the anterior midgut was a general physiological response under control of different convergent signals. Besides that, the comparison between different treatments for artificial blood feeding showed that heparinated blood was the choice with minor side effects for the study of the midgut α-L-fucosidase, when compared to defibrinated or citrated blood.

Knockdown of carnitine palmitoyltransferase I (CPT1) reduces fat body lipid mobilization and resistance to starvation in the insect vector Rhodnius prolixus.

The energy stored in fatty acids is essential for several critical activities of insects, such as embryogenesis, oviposition, and flight. Rhodnius prolixus is an obligatory hematophagous hemipteran and vector of Chagas disease, and it feeds infrequently on very large blood meals. As digestion slowly occurs, lipids are synthesized and accumulate in the fat body, mainly as triacylglycerol, in lipid droplets. Between feeding bouts, proper mobilization and oxidation of stored lipids are crucial for survival, and released fatty acids are oxidized by mitochondrial ß-oxidation. Carnitine palmitoyl transferase I (CPT1) is the enzyme that catalyzes the first reaction of the carnitine shuttle, where the activated fatty acid, acyl-CoA, is converted to acyl-carnitine to be transported into the mitochondria. Here, we investigated the role of CPT1 in lipid metabolism and in resistance to starvation in Rhodnius prolixus. The expression of the CPT1 gene (RhoprCpt1) was determined in the organs of adult females on the fourth day after a blood meal, and the flight muscle showed higher expression levels than the ovary, fat body, and anterior and posterior midgut. RhoprCpt1 expression in the fat body dramatically decreased after feeding, and started to increase again 10 days later, but no changes were observed in the flight muscle. ß-oxidation rates were determined in flight muscle and fat body homogenates with the use of 3H-palmitate, and in unfed females, they were higher in the flight muscle. In the fat body, lipid oxidation activity did not show any variation before or at different days after feeding, and was not affected by the presence of etomoxir or malonyl-CoA. We used RNAi and generated RhoprCPT1-deficient insects, which surprisingly did not show a decrease in measured 3H-palmitate oxidation rates. However, the RNAi-knockdown females presented increased amounts of triacylglycerol and larger lipid droplets in the fat body, but not in the flight muscle. When subjected to starvation, these insects had a shorter lifespan. These results indicated that the inhibition of RhoprCpt1 expression compromised lipid mobilization and affected resistance to starvation.

Alterations in energy metabolism of Rhodnius prolixus induced by Trypanosoma rangeli infection.

Trypanosoma rangeli is a protozoan parasite that infects triatomines and mammals in the Americas, producing mixed infections with Trypanosoma cruzi, the etiological agent of Chagas disease. The former parasite is not pathogenic to humans, but has different levels of pathogenicity, as well as causing physiological and behavioral alterations, to its invertebrate hosts. In this study, we measured locomotory activity, and the glyceride accumulation profile in the hemolymph and fat body, as well as the expression of key genes related to triglyceride metabolism, of Rhodnius prolixus nymphs infected with T. rangeli. We found that the locomotory activity of the insects was correlated with the amount of triglycerides in the fat body. Infected nymphs had increased activity when starved, and also had an accumulation of glycerides in the fat body and hemolymph. These alterations were also associated with a higher expression of the diacylglycerol acyltransferase, lipophorin and lipophorin receptor genes in the fat body. We infer that T. rangeli is able to alter the energetic processes of its invertebrate host, in order to increase the availability of lipids to the parasite, which, in turn modifies the activity levels of the insect. These alterations are discussed with regard to their potential to increase the transmission rate of the parasite.

A pro-BMP function exerted by Rhodnius prolixus short gastrulation reveals great diversity in the role of BMP modulators during embryonic patterning.

Dorsal-ventral (DV) patterning is regulated by the bone morphogenetic pathway (BMP) in Bilateria. In insect DV patterning, the Toll pathway also plays a role, in addition to BMPs. Variations in the relative importance of each pathway for DV patterning have been reported using single species of coleopteran, hymenopteran, hemipteran and orthopteran insects. To investigate if the molecular control of DV patterning is conserved inside an insect order, the emergent model hemiptera species Rhodnius prolixus was studied. We found that R. prolixus BMP pathway controls the entire DV axis, with a broader effect respective to Toll, as shown for the hemiptera Oncopeltus fasciatus. Different from O. fasciatus, the unique R. prolixus short gastrulation (sog) and the twisted gastrulation (tsg) orthologues do not antagonize, but rather favour embryonic BMP signalling. Our results reinforce the hypothesis that hemiptera rely preferentially on BMPs for DV patterning but that, surprisingly, in R. prolixus Sog and Tsg proteins exert only a positive role to establish a dorsal-to-ventral BMP gradient. Since sog has been reported to be lost from orthopteran and hymenopteran genomes, our results indicate that Sog's role to modify BMP activity varies greatly in different insect species.

Investigation of Rhodnius prolixus head anatomy using microtomography.

In the last years, microtomography has proved to be a powerful technique on insects' studies, allowing a detailed view of the structures' internal with a high resolution. One of the most important advantages about the use of microtomography in these studies is the fact that the dissection is not necessary, which decreases considerably the number of samples used on the insects' research. Some insects are used constantly in studies about morphology, metamorphosis, and reproduction, because they work as a model for others, and Rhodnius prolixus is one of the most studied in this group. This insect is also one of the main insect vectors of Chagas disease that kills around 12,000 people every year in Latin America. Some studies using laboratory microtomography conventional scanners combining with the correct staining methods have proved that it could be a powerful tool in biological research, allowing the visualisation of low-density tissues. The main goal of the present work was to use staining protocols to study Rhodnius prolixus with laboratory microtomography conventional scanners. The experiments were carried out at the imaging lab in the Theoretical Biology Department, University of Vienna, using an Xradia MicroXCT and at the University of Oslo, using a Skyscan 2211.

Functional characterization of maternally accumulated hydrolases in the mature oocytes of the vector Rhodnius prolixus reveals a new protein phosphatase essential for the activation of the yolk mobilization and embryo development.

Yolk biogenesis and consumption have been well conserved in oviparous animals throughout evolution. Most egg-laying animals store yolk proteins within the oocytes' yolk granules (Ygs). Following fertilization, the Ygs participate in controlled pathways of yolk breakdown to support the developing embryo's anabolic metabolism. While the unfolding of the yolk degradation program is a crucial process for successful development in many species, the molecular mechanisms responsible for yolk mobilization are still mysterious and have mostly not been explored. Here, we investigate the functional role of the oocyte maternally accumulated mRNAs of a protein phosphatase (PP501) and two aspartic proteases (cathepsin-D 405, CD405 and cathepsin-D 352, CD352) in the yolk degradation and reproduction of the insect vector of Chagas disease Rhodnius prolixus. We found that PP501 and CD352 are highly expressed in the vitellogenic ovary when compared to the other organs of the adult insect. Parental RNAi silencing of PP501 resulted in a drastic reduction in oviposition and increased embryo lethality whereas the silencing of CD352 resulted only in a slight decrease in oviposition and embryo viability. To further investigate the PP501-caused high reproduction impairment, we investigated the Ygs biogenesis during oocyte maturation and the activation of the yolk degradation program at early development. We found that the Ygs biogenesis was deficient during oogenesis, as seen by flow cytometry, and that, although the PP501-silenced unviable eggs were fertilized, the Ygs acidification and acid phosphatase activity were affected, culminating in a full impairment of the yolk proteins degradation at early embryogenesis. Altogether we found that PP501 is required for the oocyte maturation and the activation of the yolk degradation, being, therefore, essential for this vector reproduction.

Blood meal digestion and changes in lipid reserves are associated with the post-ecdysis development of the flight muscle and ovary in young adults of Rhodnius prolixus.

Rhodnius prolixus is a hemimetabolous, hematophagous insect, and both nymphs and adults feed exclusively on blood. The blood feeding triggers the molting process and, after five nymphal instar stages, the insect reaches the winged adult form. After the final ecdysis, the young adult still has a lot of blood in the midgut and, thus, we have investigated the changes in protein and lipid contents that are observed in the insect organs as digestion continues after molting. Total midgut protein content decreased during the days after the ecdysis, and digestion was finished fifteen days later. Simultaneously, proteins and triacylglycerols present in the fat body were mobilized, and their contents decreased, whereas they increased in both the ovary and the flight muscle. In order to evaluate the activity of de novo lipogenesis of each organ, the fat body, ovary and flight muscle were incubated in the presence of radiolabeled acetate, and the fat body showed the highest efficiency rate (around 47%) to convert the taken up acetate into lipids. The levels of de novo lipid synthesis in the flight muscle and ovary were very low. When 3H-palmitate was injected into the young females, its incorporation by the flight muscle was higher than by the ovary or the fat body. In the flight muscle, the 3H-palmitate was similarly distributed amongst triacylglycerols, phospholipids, diacylglycerols and free fatty acids, while in the ovary and fat body it was mostly found in triacylglycerols and phospholipids. The flight muscle was not fully developed after the molt, and at day two no lipid droplets were observed. At day five, very small lipid droplets were present, and they increased in size up to day fifteen. The diameter of the muscle fibers also increased from day two to fifteen, as well as the internuclear distance, indicating that muscle hypertrophy occurred along these days. The lipid droplets from the fat body showed a different pattern, and their diameter decreased after day two, but started to increase again at day ten. The data presented herein describes the development of the flight muscle after the final ecdysis, and modifications that occur regarding lipid stores. We show that, after molting, substrates that are present in the midgut and fat body are mobilized and directed to the ovary and flight muscle, for the adults of R. prolixus to be ready to feed and reproduce.

Topical delivery of dsRNA in two hemipteran species: Evaluation of RNAi specificity and non-target effects.

Double-stranded (ds) RNA-based technologies could provide novel and potential tool for pest management with efficiency and specificity of action. However, before applying this technique in the field, it is necessary to identify effective delivery methods and evaluate the non-target effects that may occur. In this article, we evaluated the effectiveness of dsRNA by topical delivery on a species of great agricultural interest, Halyomorpha halys. The specificity of action of the dsRNA was also investigated in Rhodnius prolixus, an insect phylogenetically close to H. halys. Of the three investigated genes (putative ATPase N2B, ATPase, serine/threonine-protein phosphatase PP1-ß catalytic subunit, PP1, and IAP repeat-containing protein 7-B-like, IAP), IAP and ATPase were able to induce higher mortality in H. halys nymphs compared to the control, with specific concentrations for each gene targeted. However, when the same RNAs were topically delivered to both R. prolixus 2nd and 3rd instar nymphs, no gene silencing and mortality were observed. For this reason, to assess dsRNA application-mediated non-target effects, we injected both H. halys and R. prolixus specific dsRNA in R. prolixus 5th instar nymphs. When the dsRNA targeting H. halys IAP was microinjected into R. prolixus 5th instar nymphs, no mortality was observed, suggesting a strong RNAi specificity. Together, these data suggest that the topical delivery could be suitable for the dsRNA to control H. halys population. Furthermore, its specificity of action would allow treatments towards single harmful species with limited non-target effects.

Laboratory Evaluation and Field Feasibility of Micro-Encapsulated Insecticide Effect on Rhodnius prolixus and Triatoma dimidiata Mortality in Rural Households in Boyacá, Colombia.

Chagas disease is a neglected vector-borne zoonosis caused by the parasite Trypanosoma cruzi that is primarily transmitted by insects of the subfamily Triatominae. Although control efforts targeting domestic infestations of Rhodnius prolixus have been largely successful, with several regions in Boyacá department certified free of T. cruzi transmission by intradomicile R. prolixus, novel native species are emerging, increasing the risk of disease. Triatoma dimidiata is the second most important species in Colombia, and conventional control methods seem to be less effective. In this study we evaluated the efficacy and usefulness of micro-encapsulated insecticide paints in laboratory conditions and its applicability in rural communities to avoid triatomine domiciliation. Laboratory conditions measured mortality at 6 months and 12 months, with an average mortality between 93-100% for T. dimidiata and 100% for R. prolixus. Evaluation of triatomine infestation in rural households was measured after one year, with an overall perception of effectiveness in reducing household domiciliation. Although triatomines were still spotted inside and around the homes, our findings demonstrate the ability of micro-encapsulated insecticide to prevent colonization inside the households when comparing infestation rates from previous years. Current control measures suggest insecticide spraying every six months, which implies great economic cost and logistical effort. Complementary triatomine control measures with insecticide spraying and micro-encapsulated insecticide paint would make public health efforts more efficient and reduce the frequency of treatment.