Genome of Rhodnius prolixus, an insect vector of Chagas disease, reveals unique adaptations to hematophagy and parasite infection.

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 immune-deficiency (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.

Glycogen Synthase Kinase-3 is involved in glycogen metabolism control and embryogenesis of Rhodnius prolixus.

Rhodnius prolixus is a blood-feeding insect that transmits Trypanosoma cruzi and Trypanosoma rangeli to vertebrate hosts. Rhodnius prolixus is also a classical model in insect physiology, and the recent availability of R. prolixus genome has opened new avenues on triatomine research. Glycogen synthase kinase 3 (GSK-3) is classically described as a key enzyme involved in glycogen metabolism, also acting as a downstream component of the Wnt pathway during embryogenesis. GSK-3 has been shown to be highly conserved among several organisms, mainly in the catalytic domain region. Meanwhile, the role of GSK-3 during R. prolixus embryogenesis or glycogen metabolism has not been investigated. Here we show that chemical inhibition of GSK-3 by alsterpaullone, an ATP-competitive inhibitor of GSK3, does not affect adult survival rate, though it alters oviposition and egg hatching. Specific GSK-3 gene silencing by dsRNA injection in adult females showed a similar phenotype. Furthermore, bright field and 4'-6-diamidino-2-phenylindole (DAPI) staining analysis revealed that ovaries and eggs from dsGSK-3 injected females exhibited specific morphological defects. We also demonstrate that glycogen content was inversely related to activity and transcription levels of GSK-3 during embryogenesis. Lastly, after GSK-3 knockdown, we observed changes in the expression of the Wingless (Wnt) downstream target ß-catenin as well as in members of other pathways such as the receptor Notch. Taken together, our results show that GSK-3 regulation is essential for R. prolixus oogenesis and embryogenesis.

Protein 2DE reference map of the anterior midgut of the blood-sucking bug Rhodnius prolixus.

Rhodnius prolixus is an important vector of Trypanosoma cruzi, the causative agent of Chagas' disease, an illness that affects 20% of Latin America population. The obligatory course of the parasite in the vector digestive tract has made it an important target for investigation in order to control the parasite transmission and thus interrupt its biological cycle in the insect vector. Therefore, an insight into the vector midgut physiology is valuable for insect control as well as to provide potential novel targets for drugs and vaccines development and thus disease treatment. In this study, the first 2DE map of R. prolixus anterior midgut is described. Proteins were separated by 2DE and analyzed by nano-LC MS/MS. The results yielded 489 proteins from 475 spots. These proteins were classified into 28 functional groups and their physiological roles in the insect midgut are discussed. All MS data have been deposited in the ProteomeXchange with identifiers PXD001488 and PXD001489 (http://proteomecentral.proteomexchange.org/dataset/PXD001488, http://proteomecentral.proteomexchange.org/dataset/PXD001489).

Ovarian dual oxidase (Duox) activity is essential for insect eggshell hardening and waterproofing.

In insects, eggshell hardening involves cross-linking of chorion proteins via their tyrosine residues. This process is catalyzed by peroxidases at the expense of H2O2 and confers physical and biological protection to the developing embryo. Here, working with Rhodnius prolixus, the insect vector of Chagas disease, we show that an ovary dual oxidase (Duox), a NADPH oxidase, is the source of the H2O2 that supports dityrosine-mediated protein cross-linking and eggshell hardening. RNAi silencing of Duox activity decreased H2O2 generation followed by a failure in embryo development caused by a reduced resistance to water loss, which, in turn, caused embryos to dry out following oviposition. Phenotypes of Duox-silenced eggs were reversed by incubation in a water-saturated atmosphere, simultaneous silencing of the Duox and catalase genes, or H2O2 injection into the female hemocoel. Taken together, our results show that Duox-generated H2O2 fuels egg chorion hardening and that this process plays an essential role during eggshell waterproofing.

Stable internal reference genes for quantitative RT-PCR analyses in Rhipicephalus microplus during embryogenesis.

Studies on the transcriptional control of gene expression are crucial to understand changes in organism's physiological or cellular conditions. To obtain reliable data on mRNA amounts and the estimation of gene expression levels, it is crucial to normalize the target gene with one or more internal reference gene(s). However, the use of constitutive genes as reference genes is controversial, as their expression patterns are sometimes more complex than previously thought. In various arthropod vectors, including ticks, several constitutive genes have been identified by studying gene expression in different tissues and life stages. The cattle tick Rhipicephalus microplus is a major vector for several pathogens and is widely distributed in tropical and subtropical regions globally. Tick developmental physiology is an essential aspect of research, particularly embryogenesis, where many important developmental events occur, thus the identification of stable reference genes is essential for the interpretation of reliable gene expression data. This study aimed to identify and select R. microplus housekeeping genes and evaluate their stability during embryogenesis. Reference genes used as internal control in molecular assays were selected based on previous studies. These genes were screened by quantitative PCR (qPCR) and tested for gene expression stability during embryogenesis. Results demonstrated that the relative stability of reference genes varied at different time points during the embryogenesis. The GeNorm tool showed that elongation factor 1α (Elf1a) and ribosomal protein L4 (Rpl4) were the most stable genes, while H3 histone family 3A (Hist3A) and ribosomal protein S18 (RpS18) were the least stable. The NormFinder tool showed that Rpl4 was the most stable gene, while the ranking of Elf1a was intermediate in all tested conditions. The BestKeeper tool showed that Rpl4 and cyclophilin A (CycA) were the more and less stable genes, respectively. These data collectively demonstrate that Rpl4, Elf1a, and GAPDH are suitable internal controls for normalizing qPCR during R. microplus embryogenesis. These genes were consistently identified as the most stable in various analysis methods employed in this study. Thus, findings presented in this study offer valuable information for the study of gene expression during embryogenesis in R. microplus.

Coxiella Endosymbiont of Rhipicephalus microplus Modulates Tick Physiology With a Major Impact in Blood Feeding Capacity.

In the past decade, metagenomics studies exploring tick microbiota have revealed widespread interactions between bacteria and arthropods, including symbiotic interactions. Functional studies showed that obligate endosymbionts contribute to tick biology, affecting reproductive fitness and molting. Understanding the molecular basis of the interaction between ticks and their mutualist endosymbionts may help to develop control methods based on microbiome manipulation. Previously, we showed that Rhipicephalus microplus larvae with reduced levels of Coxiella endosymbiont of R. microplus (CERM) were arrested at the metanymph life stage (partially engorged nymph) and did not molt into adults. In this study, we performed a transcriptomic differential analysis of the R. microplus metanymph in the presence and absence of its mutualist endosymbiont. The lack of CERM resulted in an altered expression profile of transcripts from several functional categories. Gene products such as DA-P36, protease inhibitors, metalloproteases, and evasins, which are involved in blood feeding capacity, were underexpressed in CERM-free metanymphs. Disregulation in genes related to extracellular matrix remodeling was also observed in the absence of the symbiont. Taken together, the observed alterations in gene expression may explain the blockage of development at the metanymph stage and reveal a novel physiological aspect of the symbiont-tick-vertebrate host interaction.

Neuropeptides in Rhipicephalus microplus and other hard ticks.

The synganglion is the central nervous system of ticks and, as such, controls tick physiology. It does so through the production and release of signaling molecules, many of which are neuropeptides. These peptides can function as neurotransmitters, neuromodulators and/or neurohormones, although in most cases their functions remain to be established. We identified and performed in silico characterization of neuropeptides present in different life stages and organs of Rhipicephalus microplus, generating transcriptomes from ovary, salivary glands, fat body, midgut and embryo. Annotation of synganglion transcripts led to the identification of 32 functional categories of proteins, of which the most abundant were: secreted, energetic metabolism and oxidant metabolism/detoxification. Neuropeptide precursors are among the sequences over-represented in R. microplus synganglion, with at least 5-fold higher transcription compared with other stages/organs. A total of 52 neuropeptide precursors were identified: ACP, achatin, allatostatins A, CC and CCC, allatotropin, bursicon A/B, calcitonin A and B, CCAP, CCHamide, CCRFamide, CCH/ITP, corazonin, DH31, DH44, eclosion hormone, EFLamide, EFLGGPamide, elevenin, ETH, FMRFamide myosuppressin-like, glycoprotein A2/B5, gonadulin, IGF, inotocin, insulin-like peptides, iPTH, leucokinin, myoinhibitory peptide, NPF 1 and 2, orcokinin, proctolin, pyrokinin/periviscerokinin, relaxin, RYamide, SIFamide, sNPF, sulfakinin, tachykinin and trissin. Several of these neuropeptides have not been previously reported in ticks, as the presence of ETH that was first clearly identified in Parasitiformes, which include ticks and mites. Prediction of the mature neuropeptides from precursor sequences was performed using available information about these peptides from other species, conserved domains and motifs. Almost all neuropeptides identified are also present in other tick species. Characterizing the role of neuropeptides and their respective receptors in tick physiology can aid the evaluation of their potential as drug targets.

Tigutcystatin, a cysteine protease inhibitor from Triatoma infestans midgut expressed in response to Trypanosoma cruzi.

The insect Triatoma infestans is a vector of Trypanosoma cruzi, the etiological agent of Chagas disease. A cDNA library was constructed from T. infestans anterior midgut, and 244 clones were sequenced. Among the EST sequences, an open reading frame (ORF) with homology to a cystatin type 2 precursor was identified. Then, a 288-bp cDNA fragment encoding mature cystatin (lacking signal peptide) named Tigutcystatin was cloned fused to a N-terminal His tag in pET-14b vector, and the protein expressed in Escherichia coli strain Rosetta gami. Tigutcystatin purified and cleaved by thrombin to remove His tag presented molecular mass of 11 kDa and 10,137 Da by SDS-PAGE and MALDI-TOF mass spectrometry, respectively. Purified Tigutcystatin was shown to be a tight inhibitor towards cruzain, a T. cruzi cathepsin L-like enzyme (K(i)=3.29 nM) and human cathepsin L (K(i)=3.78 nM). Tissue specific expression analysis showed that Tigutcystatin was mostly expressed in anterior midgut, although amplification in small intestine was also detected by semi quantitative RT-PCR. qReal time PCR confirmed that Tigutcystatin mRNA is significantly up-regulated in anterior midgut when T. infestans is infected with T. cruzi. Together, these results indicate that Tigutcystatin may be involved in modulation of T. cruzi in intestinal tract by inhibiting parasite cysteine proteases, which represent the virulence factors of this protozoan.

The intracellular bacterium Rickettsia rickettsii exerts an inhibitory effect on the apoptosis of tick cells.

BACKGROUND: Rickettsia rickettsii is a tick-borne obligate intracellular bacterium that causes Rocky Mountain spotted fever, a life-threatening illness. To obtain an insight into the vector-pathogen interactions, we assessed the effects of infection with R. rickettsii on the proteome cells of the tick embryonic cell line BME26. METHODS: The proteome of BME26 cells was determined by label-free high-performance liquid chromatography coupled with tandem mass spectrometry analysis. Also evaluated were the effects of infection on the activity of caspase-3, assessed by the hydrolysis of a synthetic fluorogenic substrate in enzymatic assays, and on the exposition of phosphatidyserine, evaluated by live-cell fluorescence microscopy after labeling with annexin-V. Finally, the effects of activation or inhibition of caspase-3 activity on the growth of R. rickettsii in BME26 cells was determined. RESULTS: Tick proteins of different functional classes were modulated in a time-dependent manner by R. rickettsii infection. Regarding proteins involved in apoptosis, certain negative regulators were downregulated at the initial phase of the infection (6 h) but upregulated in the middle of the exponential phase of the bacterial growth (48 h). Microorganisms are known to be able to inhibit apoptosis of the host cell to ensure their survival and proliferation. We therefore evaluated the effects of infection on classic features of apoptotic cells and observed DNA fragmentation exclusively in noninfected cells. Moreover, both caspase-3 activity and phosphatidylserine exposition were lower in infected than in noninfected cells. Importantly, while the activation of caspase-3 exerted a detrimental effect on rickettsial proliferation, its inhibition increased bacterial growth. CONCLUSIONS: Taken together, these results show that R. rickettsii modulates the proteome and exerts an inhibitory effect on apoptosis in tick cellsthat seems to be important to ensure cell colonization.

A physiologic overview of the organ-specific transcriptome of the cattle tick Rhipicephalus microplus.

To further obtain insights into the Rhipicephalus microplus transcriptome, we used RNA-seq to carry out a study of expression in (i) embryos; (ii) ovaries from partially and fully engorged females; (iii) salivary glands from partially engorged females; (iv) fat body from partially and fully engorged females; and (v) digestive cells from partially, and (vi) fully engorged females. We obtained > 500 million Illumina reads which were assembled de novo, producing > 190,000 contigs, identifying 18,857 coding sequences (CDS). Reads from each library were mapped back into the assembled transcriptome giving a view of gene expression in different tissues. Transcriptomic expression and pathway analysis showed that several genes related in blood digestion and host-parasite interaction were overexpressed in digestive cells compared with other tissues. Furthermore, essential genes for the cell development and embryogenesis were overexpressed in ovaries. Taken altogether, these data offer novel insights into the physiology of production and role of saliva, blood digestion, energy metabolism, and development with submission of 10,932 novel tissue/cell specific CDS to the NCBI database for this important tick species.

Inhibition of energy metabolism by 3-bromopyruvate in the hard tick Rhipicephalus microplus.

The cattle tick R. microplus is the biggest obstacle to livestock rearing in tropical countries. It is responsible for billions of dollars in losses every year, affecting meat and milk production, beef and dairy cattle, and the leather industry. The lack of knowledge and strategies to combat the tick only increases the losses, it leads to successive and uncontrolled applications of acaricides, favouring the selection of strains resistant to commercially available chemical treatments. In this paper, we tested 3­bromopyruvate (3­BrPA), an alkylating agent with a high affinity for cysteine residues, on the R. microplus metabolism. We found that 3-BrPA was able to induce cell death in an assay using BME26 strain cell cultures derived from embryos, it was also able to reduce cellular respiration in developing embryos. 3-BrPA is a nonspecific inhibitor, affecting enzymes of different metabolic pathways in R. microplus. In our experiments, we demonstrated that 3-BrPA was able to affect the glycolytic enzyme hexokinase, reducing its activity by approximately 50%; and it strongly inhibited triose phosphate isomerase, which is an enzyme involved in both glycolysis and gluconeogenesis. Also, the mitochondrial respiratory chain was affected, NADH cytochrome c reductase (complex I-III) and succinate cytochrome c reductase (complex II-III) were strongly inhibited by 3-BrPA. Glutamate dehydrogenase was also affected by 3-BrPA, showing a gradual inhibition of activity in all the 3-BrPA concentrations tested. Altogether, these results show that 3-BrPA is a harmful compound to the tick organism.

A novel method for measuring fructose ingestion by mosquitoes.

We adapted the Seliwanoff method to quantify fructose in mosquitoes. This method showed a minimum detection limit of 2.4 microg of fructose, and was more reliable and nearly four times more sensitive than the anthrone test. The Seliwanoffmethod was used to measure the maximum sugar intake by individual mosquitoes and to determine the digestion time of this nutrient by both Aedes aegypti and Aedes albopictus in the laboratory. Sugar intake by Ae. albopictus was up to 1.7 times higher than that of Ae. aegypti. The amount of sugar ingested by females was up to 2.5 times higher than that of males in both species. After 48 h, a fructose meal was not detected any longer in either species. The Seliwanoffmethod was applied to measure fructose content of field-collected Ae. aegypti males and females in Rio de Janeiro. Results showed that even Ae. aegypti females do feed on sugars. The standardized Seliwanoff method proved to be reliable for measuring the sugar content of individual mosquitoes and can be used wherever estimation of small quantities of fructose is needed.

Differential transcript profile of inhibitors with potential anti-venom role in the liver of juvenile and adult Bothrops jararaca snake.

BACKGROUND: Snakes belonging to the Bothrops genus are vastly distributed in Central and South America and are responsible for most cases of reported snake bites in Latin America. The clinical manifestations of the envenomation caused by this genus are due to three major activities-proteolytic, hemorrhagic and coagulant-mediated by metalloproteinases, serine proteinases, phospholipases A2 and other toxic compounds present in snake venom. Interestingly, it was observed that snakes are resistant to the toxic effects of its own and other snake's venoms. This natural immunity may occur due the absence of toxin target or the presence of molecules in the snake plasma able to neutralize such toxins. METHODS: In order to identify anti-venom molecules, we construct a cDNA library from the liver of B. jararaca snakes. Moreover, we analyzed the expression profile of four molecules-the already known anti-hemorrhagic factor Bj46a, one gamma-phospholipase A2 inhibitor, one inter-alpha inhibitor and one C1 plasma protease inhibitor-in the liver of juvenile and adult snakes by qPCR. RESULTS: The results revealed a 30-fold increase of gamma-phospholipase A2 inhibitor and a minor increase of the inter-alpha inhibitor (5-fold) and of the C1 inhibitor (3-fold) in adults. However, the Bj46a factor seems to be equally transcribed in adults and juveniles. DISCUSSION: The results suggest the up-regulation of different inhibitors observed in the adult snakes might be a physiological adaptation to the recurrent contact with their own and even other snake's venoms throughout its lifespan. This is the first comparative analysis of ontogenetic variation of expression profiles of plasmatic proteins with potential anti-venom activities of the venomous snake B. jararaca. Furthermore, the present data contributes to the understanding of the natural resistance described in these snakes.

Adaptations against heme toxicity in blood-feeding arthropods.

A blood-sucking habit appeared independently several times in the course of arthropod evolution. However, from more than a million species of insects and arachnids presently living on earth, only about 14,000 species developed the capacity to feed on vertebrate blood. This figure suggests the existence of severe physiological constraints for the evolution of hematophagy, implying the selective advantage of special adaptations related to the use of blood as a food source. Digestion of vertebrate hemoglobin in the midgut of blood-feeding arthropods results in the production of large amounts of heme, a potentially cytotoxic molecule. Here we will review mechanisms by which heme can exert biological damage, together with a wide spectrum of adaptations developed by blood-feeding insects and ticks to counteract its deleterious effects. In spite of the existence of a great molecular diversity of protective mechanisms, different hematophagous organisms developed convergent solutions that may be physiologically equivalent.

Unique PFK regulatory property from some mosquito vectors of disease, and from Drosophila melanogaster.

BACKGROUND: Arthropod-borne diseases are some of the most rapidly spreading diseases. Reducing the vector population is currently the only effective way to reduce case numbers. Central metabolic pathways are potential targets to control vector populations, but have not been well explored to this aim. The information available on energy metabolism, as a way to control lifespan and dispersion through flight of dipteran vectors, is inadequate. METHODS: Phosphofructokinase (PFK) activity was measured in the presence of both of its substrates, fructose-6-phosphate (F6P) and ATP, as well as some allosteric effectors: Fructose- 2,6 - bisphosphate (F2, 6BP), citrate and AMP. Aedes aegypti phosphofructokinase sequence (AaPFK) was aligned with many other insects and also vertebrate sequences. A 3D AaPFK model was produced and docking experiments were performed with AMP and citrate. RESULTS: The kinetic parameters of AaPFK were determined for both substrates: F6P (V = 4.47 ± 0.15 µmol of F1, 6BP/min, K0.5 = 1.48 ± 0.22 mM) and ATP (V = 4.73 ± 0.57 µmol of F1, 6BP/min, K0.5 = 0.43 ± 0.10 mM). F2,6P was a powerful activator of AaPFK, even at low ATP concentrations. AaPFK inhibition by ATP was not enhanced by citrate, consistent with observations in other insects. After examining the sequence alignment of insect and non-insect PFKs, the hypothesis is that a modification of the citrate binding site is responsible for this unique behavior. AMP, a well-known positive effector of PFK, was not capable of reverting ATP inhibition. Aedes, Anopheles and Culex are dengue, malaria and filariasis vectors, respectively, and are shown to have this distinct characteristic in phosphofructokinase control. The alignment of several insect PFKs suggested a difference in the AMP binding site and a significant change in local charges, which introduces a highly negative charge in this part of the protein, making the binding of AMP unlikely. This hypothesis was supported by 3D modeling of PFK with AMP docking, which suggested that the AMP molecule binds in a reverse orientation due to the electrostatic environment. The present findings imply a potential new way to control PFK activity and are a unique feature of these Diptera. CONCLUSIONS: The present findings provide the first molecular explanation for citrate insensitivity in insect PFKs, as well as demonstrating for the first time AMP insensitivity in dipterans. It also identified a potential target for novel insecticides for the control of arthropod-borne diseases.

Exploring the immune signalling pathway-related genes of the cattle tick Rhipicephalus microplus: From molecular characterization to transcriptional profile upon microbial challenge.

In dipteran insects, invading pathogens are selectively recognized by four major pathways, namely Toll, IMD, JNK, and JAK/STAT, and trigger the activation of several immune effectors. Although substantial advances have been made in understanding the immunity of model insects such as Drosophila melanogaster, knowledge on the activation of immune responses in other arthropods such as ticks remains limited. Herein, we have deepened our understanding of the intracellular signalling pathways likely to be involved in tick immunity by combining a large-scale in silico approach with high-throughput gene expression analysis. Data from in silico analysis revealed that although both the Toll and JAK/STAT signalling pathways are evolutionarily conserved across arthropods, ticks lack central components of the D. melanogaster IMD pathway. Moreover, we show that tick immune signalling-associated genes are constitutively transcribed in BME26 cells (a cell lineage derived from embryos of the cattle tick Rhipicephalus microplus) and exhibit different transcriptional patterns in response to microbial challenge. Interestingly, Anaplasma marginale, a pathogen that is naturally transmitted by R. microplus, causes downregulation of immune-related genes, suggesting that this pathogen may manipulate the tick immune system, favouring its survival and vector colonization.

Identification of a selenium-dependent glutathione peroxidase in the blood-sucking insect Rhodnius prolixus.

The selenium-dependent glutathione peroxidase (SeGPx) is a well-studied enzyme that detoxifies organic and hydrogen peroxides and provides cells or extracellular fluids with a key antioxidant function. The presence of a SeGPx has not been unequivocally demonstrated in insects. In the present work, we identified the gene and studied the function of a Rhodnius prolixus SeGPx (RpSeGPx). The RpSeGPx mRNA presents the UGA codon that encodes the active site selenocysteine (Sec) and a corresponding Sec insertion sequence (SECIS) in the 3' UTR region. The encoded protein includes a signal peptide, which is consistent with the high levels of GPx enzymatic activity in the insect's hemolymph, and clusters phylogenetically with the extracellular mammalian GPx03. This result contrasts with all other known insect GPxs, which use a cysteine residue instead of Sec and cluster with the mammalian phospholipid hydroperoxide GPx04. RpSeGPx is widely expressed in insect organs, with higher expression levels in the fat body. RNA interference (RNAi) was used to reduce RpSeGPx gene expression and GPx activity in the hemolymph. Adult females were apparently unaffected by RpSeGPx RNAi, whereas first instar nymphs showed a three-day delay in ecdysis. Silencing of RpSeGPx did not alter the gene expression of the antioxidant enzymes catalase, xanthine dehydrogenase and a cysteine-GPx, but it reduced the levels of the dual oxidase and NADPH oxidase 5 transcripts that encode for enzymes releasing extracellular hydrogen peroxide/superoxide. Collectively, our data suggest that RpSeGPx functions in the regulation of extracellular (hemolymph) redox homeostasis of R. prolixus.

Bioinformatic analyses of male and female Amblyomma americanum tick expressed serine protease inhibitors (serpins).

Serine protease inhibitors (serpins) are a diverse family of proteins that is conserved across taxa. The diversity of Amblyomma americanum serpins (AAS) is far more complex than previously thought as revealed by discovery of 57 and 33 AAS transcripts that are respectively expressed in male and female A. americanum ticks, with 30 found in both. While distinct reproductively, both male and female metastriate ticks, such as A. americanum, require a blood meal. Thus, 30 AAS sequences found in both male and female ticks could play important role(s) in regulating tick feeding and thus represent attractive candidates for anti-tick vaccine development. Of significant interest, 19 AAS sequences expressed in male and female ticks are also part of the 48 AAS sequences expressed in fed female tick salivary glands or midguts; two organs through which the tick interacts with host blood and immune response factors. Considered the most important domain for serpin function, the reactive center loop (RCL) is further characterized by a single 'P1' site amino acid residue, which is central to determining the protease regulated by the serpin. In this study, a diversity of 17 different P1 site amino acid residues were predicted, suggesting that A. americanum serpins potentially regulate a large number of proteolytic pathways. Our data also indicate that some serpins in this study could regulate target protease common to all tick species, in that more than 40% of AAS show 58-97% inter-species amino acid conservation. Of significance, 24% of AAS showed 62-100% inter-species conservation within the functional RCL domain, with 10 RCLs showing ≥90-100% conservation. In vertebrates, serpins with basic residues at the P1 site regulate key host defense pathways, which the tick must evade to feed successfully. Interestingly, we found that AAS sequences with basic or polar uncharged residues at the putative P1 site are more likely to be conserved across tick species. Another notable observation from our data is that AAS sequences found only in female ticks and those found in both males and females, but not those found only in male ticks, were highly conserved in other tick species. While descriptive, this study provides the basis for more in-depth studies exploring the roles of serpins in tick feeding physiology.

Non-Invasive Delivery of dsRNA into De-Waxed Tick Eggs by Electroporation.

RNA interference-mediated gene silencing was shown to be an efficient tool for validation of targets that may become anti-tick vaccine components. Here, we demonstrate the application of this approach in the validation of components of molecular signaling cascades, such as the Protein Kinase B (AKT)/Glycogen Synthase Kinase (GSK) axis during tick embryogenesis. It was shown that heptane and hypochlorite treatment of tick eggs can remove wax, affecting corium integrity and but not embryo development. Evidence of AKT and GSK dsRNA delivery into de-waxed eggs of via electroporation is provided. Primers designed to amplify part of the dsRNA delivered into the electroporated eggs dsRNA confirmed its entry in eggs. In addition, it was shown that electroporation is able to deliver the fluorescent stain, 4',6-diamidino-2-phenylindole (DAPI). To confirm gene silencing, a second set of primers was designed outside the dsRNA sequence of target gene. In this assay, the suppression of AKT and GSK transcripts (approximately 50% reduction in both genes) was demonstrated in 7-day-old eggs. Interestingly, silencing of GSK in 7-day-old eggs caused 25% reduction in hatching. Additionally, the effect of silencing AKT and GSK on embryo energy metabolism was evaluated. As expected, knockdown of AKT, which down regulates GSK, the suppressor of glycogen synthesis, decreased glycogen content in electroporated eggs. These data demonstrate that electroporation of de-waxed R. microplus eggs could be used for gene silencing in tick embryos, and improve the knowledge about arthropod embryogenesis.

A Kazal-type inhibitor is modulated by Trypanosoma cruzi to control microbiota inside the anterior midgut of Rhodnius prolixus.

The triatomine insect, Rhodnius prolixus, is a vector of Trypanosoma cruzi, a protozoan parasite that causes Chagas disease. The parasite must overcome immune response and microbiota to develop inside the midgut of triatomines. In this study, we expressed, purified and characterized a Kazal-type inhibitor from the midgut of R. prolixus, named RpTI, which may be involved in microbiota - T. cruzi interactions. The qPCR showed that the RpTI transcript was primarily expressed in tissues from the intestinal tract and that it was upregulated in the anterior midgut after T. cruzi infection. A 315-bp cDNA fragment encoding the mature protein was cloned into the pPIC9 vector and expressed in Pichia pastoris system. Recombinant RpTI (rRpTI) was purified on a trypsin-Sepharose column and had a molecular mass of 11.5 kDa as determined by SDS-PAGE analysis. This protein inhibited trypsin (Ki = 0.42 nM), whereas serine proteases from the coagulation cascade were not inhibited. Moreover, trypanocidal assays revealed that rRpTI did not interfere in the viability of T. cruzi trypomastigotes. The RpTI transcript was also knocked down by RNA interference prior to infection of R. prolixus with T. cruzi. The amount of T. cruzi in the anterior midgut was significantly lower in RpTI knockdown insects compared to the non-silenced groups. We also verified that the bacterial load is higher in the anterior midgut of silenced and infected R. prolixus compared to non-silenced and infected insects. Our results suggest that T. cruzi infection increases the expression of RpTI to mediate microbiota modulation and is important for parasite immediately after infection with R. prolixus.