Juvenile Hormone as a contributing factor in establishing midgut microbiota for fecundity and fitness enhancement in adult female Aedes aegypti.

Understanding the factors influencing mosquitoes' fecundity and longevity is important for designing better and more sustainable vector control strategies, as these parameters can impact their vectorial capacity. Here, we address how mating affects midgut growth in Aedes aegypti, what role Juvenile Hormone (JH) plays in this process, and how it impacts the mosquito's immune response and microbiota. Our findings reveal that mating and JH induce midgut growth. Additionally, the establishment of a native bacterial population in the midgut due to JH-dependent suppression of the immune response has important reproductive outcomes. Specific downregulation of AMPs with an increase in bacteria abundance in the gut results in increased egg counts and longer lifespans. Overall, these findings provide evidence of a cross-talk between JH response, gut epithelial tissue, cell cycle regulation, and the mechanisms governing the trade-offs between nutrition, immunity, and reproduction at the cellular level in the mosquito gut.

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.

Immunometabolic crosstalk in Aedes fluviatilis and Wolbachia pipientis symbiosis.

Wolbachia pipientis is a maternally transmitted symbiotic bacterium that mainly colonizes arthropods, potentially affecting different aspects of the host's physiology, e.g., reproduction, immunity, and metabolism. It has been shown that Wolbachia modulates glycogen metabolism in mosquito Aedes fluviatilis (Ae. fluviatilis). Glycogen synthesis is controlled by the enzyme GSK3, which is also involved in immune responses in both vertebrate and invertebrate organisms. Here we investigated the mechanisms behind immune changes mediated by glycogen synthase kinase ß (GSK3ß) in the symbiosis between Ae. fluviatilis and W. pipientis using a GSK3ß inhibitor or RNAi-mediated gene silencing. GSK3ß inhibition or knockdown increased glycogen content and Wolbachia population, together with a reduction in Relish2 and gambicin transcripts. Furthermore, knockdown of Relish2 or Caspar revealed that the immunodeficiency pathway acts to control Wolbachia numbers in the host. In conclusion, we describe for the first time the involvement of GSK3ß in Ae. fluviatilis immune response, acting to control the Wolbachia endosymbiotic population.

Exploring dose-response relationships in Aedes aegypti survival upon bacteria and arbovirus infection.

A detailed understanding of how host fitness changes in response to variations in microbe density (an ecological measure of disease tolerance) is an important aim of infection biology. Here, we applied dose-response curves to study Aedes aegypti survival upon exposure to different microbes. We challenged female mosquitoes with Listeria monocytogenes, a model bacterial pathogen, Dengue 4 virus and Zika virus, two medically relevant arboviruses, to understand the distribution of mosquito survival following microbe exposure. By correlating microbe loads and host health, we found that a blood meal promotes disease tolerance in our systemic bacterial infection model and that mosquitoes orally infected with bacteria had an enhanced defensive capacity than insects infected through injection. We also showed that Aedes aegypti displays a higher survival profile following arbovirus infection when compared to bacterial infections. Here, we applied a framework for investigating microbe-induced mosquito mortality and details how the lifespan of Aedes aegypti varies with different inoculum sizes of bacteria and arboviruses.

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.

The Aedes aegypti peritrophic matrix controls arbovirus vector competence through HPx1, a heme-induced peroxidase.

Aedes aegypti mosquitoes are the main vectors of arboviruses. The peritrophic matrix (PM) is an extracellular layer that surrounds the blood bolus. It acts as an immune barrier that prevents direct contact of bacteria with midgut epithelial cells during blood digestion. Here, we describe a heme-dependent peroxidase, hereafter referred to as heme peroxidase 1 (HPx1). HPx1 promotes PM assembly and antioxidant ability, modulating vector competence. Mechanistically, the heme presence in a blood meal induces HPx1 transcriptional activation mediated by the E75 transcription factor. HPx1 knockdown increases midgut reactive oxygen species (ROS) production by the DUOX NADPH oxidase. Elevated ROS levels reduce microbiota growth while enhancing epithelial mitosis, a response to tissue damage. However, simultaneous HPx1 and DUOX silencing was not able to rescue bacterial population growth, as explained by increased expression of antimicrobial peptides (AMPs), which occurred only after double knockdown. This result revealed hierarchical activation of ROS and AMPs to control microbiota. HPx1 knockdown produced a 100-fold decrease in Zika and dengue 2 midgut infection, demonstrating the essential role of the mosquito PM in the modulation of arbovirus vector competence. Our data show that the PM connects blood digestion to midgut immunological sensing of the microbiota and viral infections.

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

In triatomines, blood-feeding triggers many physiological processes including post embryonic development and reproduction. Different feeding habits, such as hematophagy, can shape gene functions to meet the challenges of each type of diet. The gut of blood-sucking insects faces particular challenges after feeding due to the quantity and the quality of the food ingested. A comparison of transcriptomic and proteomic data indicates that post transcriptional regulation of gene expression is crucial in the triatomine gut. It was proposed that eukaryotic translation initiation factor 3 subunit m (eIF3m) and eIF3e define 2 different eIF3 complexes with a distinct affinity for the different mRNAs, thus selecting the set of mRNAs to be translated and constituting a post transcriptional mode of regulation of gene expression. Because the eIF3m is mainly expressed in the gut, we evaluated its relevance in Rhodnius prolixus physiology through RNA interference-mediated gene silencing. The knockdown of eIF3m reduced the digestion rate, affecting the processes triggered by a blood meal. Its silencing inhibited molting and caused premature death in nymphs while impaired ovary development, oviposition and increased resistance to starvation in adult females. The survival of males after feeding (resistance to starvation) was not affected by eIF3m knockdown. The information regarding the eIF3m function in insects is scarce and the phenotypes observed in R. prolixus upon eIF3m silencing are different and more severe than those previously described in Drosophila melanogaster, indicating a pleiotropic role of this gene in triatomines.

The Aedes aegypti peritrophic matrix controls arbovirus vector competence through HPx1, a heme–induced peroxidase

Aedes aegypti mosquitoes are the main vectors of arboviruses. The peritrophic matrix (PM) is an extracellular layer that surrounds the blood bolus. It acts as an immune barrier that prevents direct contact of bacteria with midgut epithelial cells during blood digestion. Here, we describe a heme-dependent peroxidase, hereafter referred to as heme peroxidase 1 (HPx1). HPx1 promotes PM assembly and antioxidant ability, modulating vector competence. Mechanistically, the heme presence in a blood meal induces HPx1 transcriptional activation mediated by the E75 transcription factor. HPx1 knockdown increases midgut reactive oxygen species (ROS) production by the DUOX NADPH oxidase. Elevated ROS levels reduce microbiota growth while enhancing epithelial mitosis, a response to tissue damage. However, simultaneous HPx1 and DUOX silencing was not able to rescue bacterial population growth, as explained by increased expression of antimicrobial peptides (AMPs), which occurred only after double knockdown. This result revealed hierarchical activation of ROS and AMPs to control microbiota. HPx1 knockdown produced a 100-fold decrease in Zika and dengue 2 midgut infection, demonstrating the essential role of the mosquito PM in the modulation of arbovirus vector competence. Our data show that the PM connects blood digestion to midgut immunological sensing of the microbiota and viral infections.

Metal ions in the physiology of insects.

The 2022 Molecular Physiology volume of Current Opinion in Insect Science offers an overview of transition-metal ion (iron, copper, manganese, and zinc) biology and nonessential heavy metal ion (cadmium, lead, and methylmercury) toxicology in the model organism Drosophila melanogaster and in other insect species. An article on ticks serves as an informative evolutionary comparator for iron and heme physiology. The complex interface between environmental exposure to metals, symbiotic or pathogenic microbes, and insect behavior and reproduction is considered.

Wolbachia pipientis modulates metabolism and immunity during Aedes fluviatilis oogenesis.

Wolbachia pipientis is a maternally transmitted bacterium that mostly colonizes arthropods, including the mosquito Aedes fluviatilis, potentially affecting different aspects of host physiology. This intracellular bacterium prefers gonadal tissue cells, interfering with the reproductive cycle of insects, arachnids, crustaceans, and nematodes. Wolbachia's ability to modulate the host's reproduction is related to its success in prevalence and frequency. Infecting oocytes is essential for vertical propagation, ensuring its presence in the germline. The mosquito Ae. fluviatilis is a natural host for this bacterium and therefore represents an excellent experimental model in the effort to understand host-symbiont interactions and the mutual metabolic regulation. The aim of this study was to comparatively describe metabolic changes in naturally Wolbachia-infected and uninfected ovaries of Ae. fluviatilis during the vitellogenic period of oogenesis, thus increasing the knowledge about Wolbachia parasitic/symbiotic mechanisms.

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.

Atypical strategies for cuticle pigmentation in the blood-feeding hemipteran Rhodnius prolixus.

Pigmentation in insects has been linked to mate selection and predator evasion, thus representing an important aspect for natural selection. Insect body color is classically associated to the activity of tyrosine pathway enzymes, and eye color to pigment synthesis through the tryptophan and guanine pathways, and their transport by ATP-binding cassette proteins. Among the hemiptera, the genetic basis for pigmentation in kissing bugs such as Rhodnius prolixus, that transmit Chagas disease to humans, has not been addressed. Here, we report the functional analysis of R. prolixus eye and cuticle pigmentation genes. Consistent with data for most insect clades, we show that knockdown for yellow results in a yellow cuticle, while scarlet and cinnabar knockdowns display red eyes as well as cuticle phenotypes. In addition, tyrosine pathway aaNATpreto knockdown resulted in a striking dark cuticle that displays no color pattern or UV reflectance. In contrast, knockdown of ebony and tan, that encode N-beta-alanyl dopamine hydroxylase branch tyrosine pathway enzymes, did not generate the expected dark and light brown phenotypes, respectively, as reported for other insects. We hypothesize that R. prolixus, which requires tyrosine pathway enzymes for detoxification from the blood diet, evolved an unusual strategy for cuticle pigmentation based on the preferential use of a color erasing function of the aaNATpreto tyrosine pathway branch. We also show that genes classically involved in the generation and transport of eye pigments regulate red body color in R. prolixus. This is the first systematic approach to identify the genes responsible for the generation of color in a blood-feeding hemiptera, providing potential visible markers for future transgenesis.

Beyond the eye: Kynurenine pathway impairment causes midgut homeostasis dysfunction and survival and reproductive costs in blood-feeding mosquitoes.

Insect ommochrome biosynthesis pathways metabolize tryptophan to generate eye-color pigments and wild-type alleles of pathway genes are useful phenotypic markers in transgenesis studies. Pleiotropic effects of mutations in some genes exert a load on both survival and reproductive success in blood-feeding species. Here, we investigated the challenges imposed on mosquitoes by the increase of tryptophan metabolites resulting from blood meal digestion and the impact of disruptions of the ommochrome biosynthesis pathway. Female mosquitoes with spontaneous and induced mutations in the orthologs of the genes encoding kynurenine hydroxylase in Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus exhibited impaired survival and reproductive phenotypes that varied in type and severity among the species. A compromised midgut permeability barrier function was also observed in An. stephensi. Surprisingly, mutant mosquitoes displayed an increase in microbiota compared to controls that was not accompanied by a general induction of immune genes. Antibiotic treatment rescued some deleterious traits implicating a role for the kynurenine pathway (KP) in midgut homeostasis. Supplemental xanthurenic acid, a KP end-product, rescued lethality and limited microbiota proliferation in Ae. aegypti. These data implicate the KP in the regulation of the host/microbiota interface. These pleiotropic effects on mosquito physiology are important in the development of genetic strategies targeting vector mosquitoes.

Delivery of Double-Stranded RNAs (dsRNAs) Produced by Escherichia coli HT115(DE3) for Nontransgenic RNAi-Based Insect Pest Management.

RNA interference (RNAi) is a powerful mechanism that can be exploited not only for physiology research but also for designing insect pest management approaches. Some insects cause harm by vectoring diseases dangerous to humans, livestock, or plants or by damaging crops. For at least a decade now, different insect control strategies that induce RNAi by delivering double stranded RNA (dsRNA) targeting essential genes have been proposed. Here, we focus on nontransgenic RNAi-based approaches that use oral delivery of dsRNA through feeding of inactivated bacteria to produce RNAi in disease vectors and in a crop pest. This potential pest management method could be easily adapted to target different genes or similar organisms.

Higher expression of proline dehydrogenase altered mitochondrial function and increased Trypanosoma cruzi differentiation in vitro and in the insect vector.

The pathogenic protist Trypanosoma cruzi uses kissing bugs as invertebrate hosts that vectorize the infection among mammals. This parasite oxidizes proline to glutamate through two enzymatic steps and one nonenzymatic step. In insect vectors, T. cruzi differentiates from a noninfective replicating form to nonproliferative infective forms. Proline sustains this differentiation, but to date, a link between proline metabolism and differentiation has not been established. In T. cruzi, the enzymatic steps of the proline-glutamate oxidation pathway are catalyzed exclusively by the mitochondrial enzymes proline dehydrogenase [TcPRODH, EC: 1.5.5.2] and Δ1-pyrroline-5-carboxylate dehydrogenase [TcP5CDH, EC: 1.2.1.88]. Both enzymatic steps produce reducing equivalents that are able to directly feed the mitochondrial electron transport chain (ETC) and thus produce ATP. In this study, we demonstrate the contribution of each enzyme of the proline-glutamate pathway to ATP production. In addition, we show that parasites overexpressing these enzymes produce increased levels of H2O2, but only those overexpressing TcP5CDH produce increased levels of superoxide anion. We show that parasites overexpressing TcPRODH, but not parasites overexpressing TcP5CDH, exhibit a higher rate of differentiation into metacyclic trypomastigotes in vitro. Finally, insect hosts infected with parasites overexpressing TcPRODH showed a diminished parasitic load but a higher percent of metacyclic trypomastigotes, when compared with controls. Our data show that parasites overexpressing both, PRODH and P5CDH had increased mitochondrial functions that orchestrated different oxygen signaling, resulting in different outcomes in relation to the efficiency of parasitic differentiation in the invertebrate host.

"Urate and NOX5 Control Blood Digestion in the Hematophagous Insect Rhodnius prolixus".

Low levels of reactive oxygen species (ROS) are now recognized as essential players in cell signaling. Here, we studied the role of two conserved enzymes involved in redox regulation that play a critical role in the control of ROS in the digestive physiology of a blood-sucking insect, the kissing bug Rhodnius prolixus. RNAi-mediated silencing of RpNOX5 and RpXDH induced early mortality in adult females after a blood meal. Recently, a role for RpNOX5 in gut motility was reported, and here, we show that midgut peristalsis is also under the control of RpXDH. Together with impaired peristalsis, silencing either genes impaired egg production and hemoglobin digestion, and decreased hemolymph urate titers. Ultrastructurally, the silencing of RpNOX5 or RpXDH affected midgut cells, changing the cells of blood-fed insects to a phenotype resembling the cells of unfed insects, suggesting that these genes work together in the control of blood digestion. Injection of either allopurinol (an XDH inhibitor) or uricase recapitulated the gene silencing effects, suggesting that urate itself is involved in the control of blood digestion. The silencing of each of these genes influenced the expression of the other gene in a complex way both in the unfed state and after a blood meal, revealing signaling crosstalk between them that influences redox metabolism and nitrogen excretion and plays a central role in the control of digestive physiology.

Non-immune Traits Triggered by Blood Intake Impact Vectorial Competence.

Blood-feeding arthropods are considered an enormous public health threat. They are vectors of a plethora of infectious agents that cause potentially fatal diseases like Malaria, Dengue fever, Leishmaniasis, and Lyme disease. These vectors shine due to their own physiological idiosyncrasies, but one biological aspect brings them all together: the requirement of blood intake for development and reproduction. It is through blood-feeding that they acquire pathogens and during blood digestion that they summon a collection of multisystemic events critical for vector competence. The literature is focused on how classical immune pathways (Toll, IMD, and JAK/Stat) are elicited throughout the course of vector infection. Still, they are not the sole determinants of host permissiveness. The dramatic changes that are the hallmark of the insect physiology after a blood meal intake are the landscape where a successful infection takes place. Dominant processes that occur in response to a blood meal are not canonical immunological traits yet are critical in establishing vector competence. These include hormonal circuitries and reproductive physiology, midgut permeability barriers, midgut homeostasis, energy metabolism, and proteolytic activity. On the other hand, the parasites themselves have a role in the outcome of these blood triggered physiological events, consistently using them in their favor. Here, to enlighten the knowledge on vector-pathogen interaction beyond the immune pathways, we will explore different aspects of the vector physiology, discussing how they give support to these long-dated host-parasite relationships.

Repurposing the orphan drug nitisinone to control the transmission of African trypanosomiasis.

Tsetse transmit African trypanosomiasis, which is a disease fatal to both humans and animals. A vaccine to protect against this disease does not exist so transmission control relies on eliminating tsetse populations. Although neurotoxic insecticides are the gold standard for insect control, they negatively impact the environment and reduce populations of insect pollinator species. Here we present a promising, environment-friendly alternative to current insecticides that targets the insect tyrosine metabolism pathway. A bloodmeal contains high levels of tyrosine, which is toxic to haematophagous insects if it is not degraded and eliminated. RNA interference (RNAi) of either the first two enzymes in the tyrosine degradation pathway (tyrosine aminotransferase (TAT) and 4-hydroxyphenylpyruvate dioxygenase (HPPD)) was lethal to tsetse. Furthermore, nitisinone (NTBC), an FDA-approved tyrosine catabolism inhibitor, killed tsetse regardless if the drug was orally or topically applied. However, oral administration of NTBC to bumblebees did not affect their survival. Using a novel mathematical model, we show that NTBC could reduce the transmission of African trypanosomiasis in sub-Saharan Africa, thus accelerating current disease elimination programmes.

Rhodnius prolixus uses the peptidoglycan recognition receptor rpPGRP-LC/LA to detect Gram-negative bacteria and activate the IMD pathway.

Insects rely on an innate immune system to recognize and eliminate pathogens. Key components of this system are highly conserved across all invertebrates. To detect pathogens, insects use Pattern recognition receptors (PRRs) that bind to signature motifs on the surface of pathogens called Pathogen Associated Molecular Patterns (PAMPs). In general, insects use peptidoglycan recognition proteins (PGRPs) in the Immune Deficiency (IMD) pathway to detect Gram-negative bacteria, and other PGRPs and Gram-negative binding proteins (GNBPs) in the Toll pathway to detect Gram-positive bacteria and fungi, although there is crosstalk and cooperation between these and other pathways. Once pathogens are recognized, these pathways activate the production of potent antimicrobial peptides (AMPs). Most PRRs in insects have been reported from genome sequencing initiatives but few have been characterized functionally. The initial studies on insect PRRs were done using established dipteran model organisms such as Drosophila melanogaster, but there are differences in the numbers and functional role of PRRs in different insects. Here we describe the genomic repertoire of PGRPs in Rhodnius prolixus, a hemimetabolous hemipteran vector of the parasite Trypanosoma cruzi that causes Chagas disease in humans. Using a de novo transcriptome from the fat body of immune activated insects, we found 5 genes encoding PGRPs. Phylogenetic analysis groups R. prolixus PGRPs with D. melanogaster PGRP-LA, which is involved in the IMD pathway in the respiratory tract. A single R. prolixus PGRP gene encodes isoforms that contain an intracellular region or motif (cryptic RIP Homotypic Interaction Motif-cRHIM) that is involved in the IMD signaling pathway in D. melanogaster. We characterized and silenced this gene using RNAi and show that the PGRPs that contain cRHIMs are involved in the recognition of Gram-negative bacteria, and activation of the IMD pathway in the fat body of R. prolixus, similar to the PGRP-LC of D. melanogaster. This is the first functional characterization of a PGRP containing a cRHIM motif that serves to activate the IMD pathway in a hemimetabolous insect.

An insight into the functional role of antioxidant and detoxification enzymes in adult Rhipicephalus microplus female ticks.

Ticks have developed physiological adaptations to transport, store, metabolize and secrete toxic components from the diet and environment. Different classes of enzymes are involved in these processes, however, the role of several of them is not yet characterized in Rhipicephalus microplus. In this context, this work investigated the action of antioxidant and detoxification enzymes, as well as the levels of essential cellular reductants in R. microplus partially engorged females (PEF) and fully engorged females (FEF). Results demonstrated that enzymes transcriptional levels and enzymatic activity from ovary and fat body were higher in PEF than in FEF, except for ovary Glutathione peroxidase (GPx), which was the only enzyme showing highest activity in the FEF stage. These results indicated a higher demand for antioxidant potential in these organs at the initial feeding phase than during egg-laying. In midgut, however, there was more variability in the transcriptional levels and activity of the different enzymes between the PEF and FEF phases. Similar NADPH levels were found in PEF and FEF phases, suggesting a remarkable capacity to maintain a regular supply of reducing power, despite the developmental changes and large intake of heme and iron. However, reduced glutathione (GSH) levels were variable between PEF and FEF when distinct organs were compared. Taken together, our data suggest a higher demand for reducing potential in FEF ticks. The silencing of catalase (CAT) or thioredoxin reductase (TRx) genes in females did not impair feeding, egg-laying capacity, or larvae hatching. CAT-silenced ticks had increased ovary peroxidase activity, a possible compensatory antioxidant mechanism. Altogether, the results shed light on the complexity of the antioxidant and detoxification enzyme system in ticks and its involvement in different physiological mechanisms.