The role of LuloPAT amino acid/proton symporters in midgut alkalinization in the sandfly Lutzomyia longipalpis (Diptera - Psychodidae).

In Lutzomyia longipalpis females, which are the main vectors of Leishmania infantum in the Americas, hematophagy is crucial for ovary development. The control of pH in the midgut during blood digestion is important to the functioning of the digestive enzymes, which release amino acids in the luminal compartment that are then transported through the enterocytes to the hemolymph for delivery to the ovary and other organs. In the present work, we investigated transport systems known as LuloPATs that are present in the midgut of L. longipalpis but not in other organs. These transporters achieve symport of amino acids with H+ ions, and one of them (LuloPAT1) is orthologous to a transporter described in Aedes aegypti. According to our results, the transcription levels of LuloPAT1 increased significantly immediately after a blood meal. Based on the variation of the fluorescence of fluorescein with the pH of the medium, we developed a technique that shows the acidification of the cytoplasm of gut cells when amino acids are cotransported with H+ from the lumen into the enterocytes. In our experiments, the midguts of the sandflies were dissected and opened longitudinally so that added amino acids could enter the enterocytes via the lumen (PAT carriers are apical). LuloPAT1 transporters are part of a complex of mechanisms that act synergistically to promote gut alkalinization as soon as blood intake by the vector occurs. In dissected but not longitudinally opened midguts, added amino acids could only enter through the basolateral region of enterocytes. However, alkalinization of the lumen was observed because the entry of some amino acids into the cytoplasm of enterocytes triggers a luminal alkalinization mechanism independent of LuloPATs. These findings provide new perspectives that will enable the characterization of the set of signaling pathways involved in pH regulation within the L. longipalpis midgut.

RNA-seq analysis of the salivary glands and midgut of the Argasid tick Ornithodoros rostratus.

Ornithodoros rostratus is a South American argasid tick which importance relies on its itchy bite and potential as disease vector. They feed on a wide variety of hosts and secrete different molecules in their saliva and intestinal content that counteract host defences and help to accommodate and metabolize the relatively large quantity of blood upon feeding. The present work describes the transcriptome profile of salivary gland (SG) and midgut (MG) of O. rostratus using Illumina sequencing. A total of 8,031 contigs were assembled and assigned to different functional classes. Secreted proteins were the most abundant in the SG and accounted for ~67% of all expressed transcripts with contigs with identity to lipocalins and acid tail proteins being the most representative. On the other hand, immunity genes were upregulated in MG with a predominance of defensins and lysozymes. Only 10 transcripts in SG and 8 in MG represented ~30% of all RNA expressed in each tissue and one single contig (the acid tail protein ORN-9707) represented ~7% of all expressed contigs in SG. Results highlight the functional difference of each organ and identified the most expressed classes and contigs of O. rostratus SG and MG.

Countercurrent heat exchange and thermoregulation during blood-feeding in kissing bugs.

Blood-sucking insects experience thermal stress at each feeding event on endothermic vertebrates. We used thermography to examine how kissing-bugs Rhodnius prolixus actively protect themselves from overheating. During feeding, these bugs sequester and dissipate the excess heat in their heads while maintaining an abdominal temperature close to ambient. We employed a functional-morphological approach, combining histology, µCT and X-ray-synchrotron imaging to shed light on the way these insects manage the flow of heat across their bodies. The close alignment of the circulatory and ingestion systems, as well as other morphological characteristics, support the existence of a countercurrent heat exchanger in the head of R. prolixus, which decreases the temperature of the ingested blood before it reaches the abdomen. This kind of system has never been described before in the head of an insect. For the first time, we show that countercurrent heat exchange is associated to thermoregulation during blood-feeding.

Functional aspects of salivary nitric oxide synthase of Rhodnius prolixus (Hemiptera, Reduviidae) and nitric oxide trafficking at the vector-host interface.

Rhodnius prolixus expresses nitric oxide synthase (NOS) in the cytosol of the salivary gland (SG) cells. The NO produced is stored in the SG lumen bound to NO-carrier haemeproteins called nitrophorins (NPs). NPs bind tightly to NO in the acidic SG lumen, but release NO when the pH becomes high, e.g., at the host skin (pH~7.4). NO elicits potent and transient relaxation of vascular smooth muscle. Here, we investigated the role of salivary NO in the R. prolixus feeding behaviour and the salivary vasodilator activity of the host microcirculation. NOS knockdown in R. prolixus changed the SG colour, decreased the number of NO-loaded NPs and caused impairment of feeding performance. When salivary gland extracts (SGEs) were obtained from NOS- and NPs-knockdown insects and prepared in pH 5.0 solution and injected (i.v.) into mice via the tail vein, no vasodilation was observed, whereas SGEs from control insects caused long-term venodilation in the mouse skin. SGs disrupted directly in PBS (pH 7.4) containing BSA produced long-term vasodilation compared to the controls without BSA due to the possible formation of nitroso-albumin, suggesting that host serum albumin extends the NO half-life when NO is injected into the host skin by triatomine during their blood-feeding.

Functional evaluation of Heat Shock Proteins 70 (HSP70/HSC70) on Rhodnius prolixus (Hemiptera, Reduviidae) physiological responses associated with feeding and starvation.

Blood-sucking vectors must overcome thermal stress caused by intake of proportionally large amounts of warm blood from their hosts. In response to this, Heat Shock Proteins (HSPs) such as the widely studied HSP70 family (the inducible HSP70 and the cognate form HSC70, known for their role in preserving essential cellular functions) are rapidly up-regulated in their tissues. The triatomine Rhodnius prolixus is an important vector of Trypanosoma cruzi, the causative pathogen of Chagas' disease, and is also a model organism for studying insect biology and physiology. In this work, we observed that the expression of Rhodnius prolixus HSP70 was rapidly up-regulated in response to thermal shocks (0 °C and 40 °C) and also during the first hours after feeding on blood. HSP70/HSC70 RNAi knockdown elicited important alterations in R. prolixus physiological responses triggered by blood meal and starvation. HSP70/HSC70 knockdown insects showed lower resistance to prolonged starvation in comparison to appropriate controls, dying between 32 and 40 days after dsRNA injection. After blood feeding, the physiological effects of HSP70/HSC70 knockdown were more prominent and the insects died even earlier, within 14-20 days after feeding (21-27 days after dsRNA injection). These bugs showed impaired blood processing and digestion, reduced energetic metabolism and the midgut immune responses were compromised. Our findings suggest that HSP70/HSC70 depletion affected R. prolixus in starvation or fed conditions. After feeding, the arrival of blood in the digestive tract of knockdown insects fails to activate essential signaling pathways involved in blood processing, producing several alterations in their physiological processes enough to generate a premature death.

Colonization of Rhodnius prolixus gut by Trypanosoma cruzi involves an extensive parasite killing.

Trypanosoma cruzi, the etiological agent of Chagas disease, is ingested by triatomines during their bloodmeal on an infected mammal. Aiming to investigate the development and differentiation of T. cruzi inside the intestinal tract of Rhodnius prolixus at the beginning of infection we fed insects with cultured epimastigotes and blood trypomastigotes from infected mice to determine the amount of recovered parasites after ingestion. Approximately 20% of the ingested parasites was found in the insect anterior midgut (AM) 3 h after feeding. Interestingly, a significant reduction (80%) in the numbers of trypomastigotes was observed after 24 h of infection suggesting that parasites were killed in the AM. Moreover, few parasites were found in that intestinal portion after 96 h of infection. The evaluation of the numbers of parasites in the posterior midgut (PM) at the same periods showed a reduced parasite load, indicating that parasites were not moving from the AM. Additionally, incubation of blood trypomastigotes with extracts from R. prolixus AMs revealed that components of this tissue could induce significant death of T. cruzi. Finally, we observed that differentiation from trypomastigotes to epimastigotes is not completed in the AM; instead we suggest that trypomastigotes change to intermediary forms before their migration to the PM, where differentiation to epimastigotes takes place. The present work clarifies controversial points concerning T. cruzi development in insect vector, showing that parasite suffers a drastic decrease in population size before epimastigonesis accomplishment in PM.

Long-term effects and parental RNAi in the blood feeder Rhodnius prolixus (Hemiptera; Reduviidae).

RNA interference (RNAi) has been widely employed as a useful alternative to study gene function in insects, including triatomine bugs. However, several aspects related to the RNAi mechanism and functioning are still unclear. The aim of this study is to investigate the persistence and the occurrence of systemic and parental RNAi in the triatomine bug Rhodnius prolixus. For such, the nitrophorins 1 to 4 (NP1-4), which are salivary hemeproteins, and the rhodniin, an intestinal protein, were used as targets for RNAi. The dsRNA for both molecules were injected separately into 3rd and 5th instar nymphs of R. prolixus and the knockdown (mRNA levels and phenotype) were progressively evaluated along several stages of the insect's life. We observed that the NP1-4 knockdown persisted for more than 7 months after the dsRNA injection, and at least 5 months in rhodniin knockdown, passing through various nymphal stages until the adult stage, without continuous input of dsRNA. The parental RNAi was successful from the dsRNA injection in 5th instar nymphs for both knockdown targets, when the RNAi effects (mRNA levels and phenotype) were observed at least in the 2nd instar nymphs of the F1 generation. However, the parental RNAi did not occur when the dsRNA was injected in the 3rd instars. The confirmation of the long persistence and parental transmission of RNAi in R. prolixus can improve and facilitate the utilization of this tool in insect functional genomic studies.

Application of RNA interference in triatomine (Hemiptera: Reduviidae) studies.

Triatomines (Hemiptera: Reduviidae) are obligate hematophagous insects. They are of medical importance because they are vectors of Trypanosoma cruzi, the causative agent of Chagas disease in the Americas. In recent years, the RNA interference (RNAi) technology has emerged as a practical and useful alternative means of studying gene function in insects, including triatomine bugs. RNAi research in triatomines is still in its early stages, several issues still need to be elucidated, including the description of the molecules involved in the RNAi machinery and aspects related to phenotype evaluation and persistence of the knockdown in different tissues and organs. This review considers recent applications of RNAi to triatomine research, describing the major methods that have been applied during the knockdown process such as the double-stranded RNA delivery mechanism (injection, microinjection, or ingestion) and the phenotype characterization (mRNA and target protein levels) in studies conducted with the intent to provide greater insights into the biology of these insects. In addition to the characterization of insect biomolecules, some with biopharmacological potential, RNAi may provide a new view of the interaction between triatomine and trypanosomatids, enabling the development of new measures for vector control and transmission of the parasite.

The interaction between Trypanosoma rangeli and the nitrophorins in the salivary glands of the triatomine Rhodnius prolixus (Hemiptera; Reduviidae).

The parasite Trypanosoma rangeli develops in the intestinal tract of triatomines and, particularly in species of the genus Rhodnius, invades the hemolymph and salivary glands, where subsequent metacyclogenesis takes place. Many aspects of the interaction between T. rangeli and triatomines are still unclear, especially concerning the development of the parasite in the salivary glands and how the parasite interacts with the saliva. In this work, we describe new findings on the process of T. rangeli infection of the salivary glands and the impact of infection on the saliva composition. To ensure a complete infection (intestinal tract, hemolymph and salivary glands), 3rd instar Rhodnius prolixus nymphs were fed on blood containing T. rangeli epimastigotes using an artificial feeder. After molt to the 4th instar, the nymphs were inoculated with epimastigotes in the hemolymph. The results showed that the flagellates started to invade the salivary glands by the 7th day after the injection. The percentage of trypomastigotes inside the salivary glands continuously increased until the 25th day, at which time the trypomastigotes were more than 95% of the T. rangeli forms present. The salivary contents from T. rangeli-infected insects showed a pH that was significantly more acidic (<6.0) and had a lower total protein and hemeprotein contents compared with non-infected insects. However, the ratio of hemeprotein to total protein was similar in both control and infected insects. qPCR demonstrated that the expression levels of three housekeeping genes (18S rRNA, ß-actin and α-tubulin) and nitrophorins 1-4 were not altered in the salivary glands after an infection with T. rangeli. In addition, the four major nitrophorins (NPs 1-4) were knocked down using RNAi and their suppression impacted T. rangeli survival in the salivary glands to the point that the parasite burden inside the R. prolixus salivary glands was reduced by more than 3-fold. These results indicated that these parasites most likely non-specifically incorporated the proteins that were present in R. prolixus saliva as nutrients, without impairing the biosynthesis of the antihemostatic molecules.

Validation of reference genes for expression analysis in the salivary gland and the intestine of Rhodnius prolixus (Hemiptera, Reduviidae) under different experimental conditions by quantitative real-time PCR.

BACKGROUND: Rhodnius prolixus is a blood-feeding insect that can transmit Trypanosoma cruzi and Trypanosoma rangeli to vertebrate hosts. Recently, genomic resources for invertebrate vectors of human pathogens have increased significantly, and R. prolixus has been one of the main species studied among the triatomines. However, the paucity of information on many of the fundamental molecular aspects of this species limits the use of the available genomic information. The present study aimed to facilitate gene expression studies by identifying the most suitable reference genes for the normalization of mRNA expression data from qPCR. RESULTS: The expression stability of five candidate reference genes (18S rRNA, GAPDH, ß-actin, α-tubulin and ribosomal protein L26) was evaluated by qPCR in two tissues (salivary gland and intestine) and under different physiological conditions: before and after blood feeding and after infection with T. cruzi or T. rangeli. The results were analyzed with three software programs: geNorm, NormFinder and BestKeeper. All of the evaluated candidate genes proved to be acceptable as reference genes, but some were found to be more appropriate depending on the experimental conditions. 18S, GAPDH and α-tubulin showed acceptable stability for studies in all of the tissues and experimental conditions evaluated. ß-actin, one of the most widely used reference genes, was confirmed to be one of the most suitable reference genes in studies with salivary glands, but it had the lowest expression stability in the intestine after insect blood feeding. L26 was identified as the poorest reference gene in the studies performed. CONCLUSIONS: The expression stability of the genes varies in different tissue samples and under different experimental conditions. The results provided by three statistical packages emphasize the suitability of all five of the tested reference genes in both the crop and the salivary glands with a few exceptions. The results emphasise the importance of validating reference genes for qRT-PCR analysis in R. prolixus studies.

Influence of the intestinal anticoagulant in the feeding performance of triatomine bugs (Hemiptera; Reduviidae).

Triatomines are haematophagous insects in all post-embryonic life stages. They are vectors of Trypanosoma cruzi, the causative agent of Chagas disease. Their vectorial ability is influenced by their feeding performance, which varies greatly amongst species. Recent work showed that inhibition of the coagulation process in the anterior midgut (crop) environment considerably influences the blood meal size. In this work, we performed a comparative study of the level of anticoagulant activity in the saliva and crop contents of three triatomine species -Triatoma infestans, Triatoma brasiliensis and Rhodnius prolixus - and correlated this with their feeding performance on live hosts. Moreover, the feeding parameters on a large diameter vessel influenced by the crop anticoagulants were evaluated in detail. The anticoagulant activity was significantly higher in the crop contents than in salivary glands, varying from 1.6-fold higher for R. prolixus to 70-fold higher for T. brasiliensis. Amongst the species, T. brasiliensis had the lowest crop anticoagulant activity, the lowest concentration of thrombin inhibitor, and took the longest to feed. Triatoma brasiliensis nymphs that had their intestinal anticoagulant (brasiliensin) knocked down by RNA interference had the lowest capacity to maintain cibarial pump frequency at higher levels throughout the feeding process and consequently a lower ingestion rate (mg/min), even when fed under favourable conditions (large diameter vessel). However, the feeding difficulty for brasiliensin knockdown T. brasiliensis nymphs was reversed by treating the host mice with heparin (a potent systemic anticoagulant) before blood feeding. The results indicate that crop anticoagulant activity influences modulation of the blood-pumping frequency to the intestine and significantly affects the feeding efficiency of triatomine spp. on live hosts.

The role of salivary nitrophorins in the ingestion of blood by the triatomine bug Rhodnius prolixus (Reduviidae: Triatominae).

To assist haematophagy, Rhodnius prolixus produces several bioactive molecules in its saliva which it injects into the host skin. The most abundant of these molecules are the nitrophorins (NPs). In this work, we reduced the expression of NP1-4 in the saliva of R. prolixus by RNAi and evaluated the subsequent feeding performance of the bugs using the cibarial pump electromyogram either on the dorsal skin or on the tail vein of the mice. NPs salivary mRNA was reduced by >99% in comparison to controls. Saliva from knockdown nymphs also presented 82% less haemproteins while the total protein was not reduced. Knockdown nymphs feeding on the skin had lower ingestion rates mainly due to the longer cumulative probing time and lower cibarial pump frequency. Another difference was that knockdown insects bit approximately 5 times more. No differences were observed between groups fed on the tail vein. When the feeding sites were compared, nymphs fed on the tail vein had higher effective ingestion rates. These findings endorse the importance of the NPs for the ability of bugs to complete the meal in a short total contact time with a low number of bites, decreasing the perception of the insect by the host.