Immunometabolic crosstalk in Aedes fluviatilis 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 GSK3ß in the symbiosis between Ae. fluviatilis and Wolbachia 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 (REL2) and gambicin transcripts. Furthermore, knockdown of REL2 or Caspar revealed that the Imd pathway acts to control Wolbachia numbers in the host. In conclusion, we describe for the first time the involvement of GSK3ß in Ae. fluviatillis immune response, acting to control the Wolbachia endosymbiotic population.

Cross-species immunoprotective antigens (subolesin, ferritin 2 and P0) provide protection against Rhipicephalus sanguineus sensu lato.

BACKGROUND: Tick control is mostly hampered by the rise of acaricide-resistant tick populations. Significant efforts have focused on developing alternative control methods, including cross-species protective and/or cocktail-based anti-tick vaccines, to achieve protection against various tick species. METHODS: In this study, full-length open reading frames encoding subolesin (SUB) from Rhipicephalus microplus and ferritin 2 (FER2) from Hyalomma anatolicum as well as the partial 60S acidic ribosomal protein (P0) from R. microplus were cloned, expressed in Escherichia coli and used as vaccine antigens against Rhipicephalus sanguineus sensu lato (R. sanguineus s.l.) infestation in rabbits. RESULTS: In silico analyses revealed that the SUB, P0 and FER2 proteins were antigenic and displayed limited similarity to the host's homologous proteins. The proteins shared identities of 97.5%, 100% and 89.5% with their SUB, P0 and FER2 R. sanguineus s.l. orthologous sequences, respectively. Antibodies against each recombinant protein cross-recognized the native proteins in the different tissues and developmental stages of R. sanguineus s.l. Overall efficacy of the SUB, FER2 and cocktail (SUB+FER2+P0) vaccines against R. sanguineus s.l. infestation was 86.3%, 95.9% and 90.9%, respectively. CONCLUSIONS: Both mono-antigen and the cocktail anti-tick vaccines affected the biological parameters of R. sanguineus s.l. infestation in the rabbit model, which could be extrapolated to its infested host under natural conditions. These findings support the possibility of using mono-antigenic and cocktail-based vaccines for large-scale anti-tick vaccine development against multiple tick species.

G6PDH as a key immunometabolic and redox trigger in arthropods.

The enzyme glucose-6-phosphate dehydrogenase (G6PDH) plays crucial roles in glucose homeostasis and the pentose phosphate pathway (PPP), being also involved in redox metabolism. The PPP is an important metabolic pathway that produces ribose and nicotinamide adenine dinucleotide phosphate (NADPH), which are essential for several physiologic and biochemical processes, such as the synthesis of fatty acids and nucleic acids. As a rate-limiting step in PPP, G6PDH is a highly conserved enzyme and its deficiency can lead to severe consequences for the organism, in particular for cell growth. Insufficient G6PDH activity can lead to cell growth arrest, impaired embryonic development, as well as a reduction in insulin sensitivity, inflammation, diabetes, and hypertension. While research on G6PDH and PPP has historically focused on mammalian models, particularly human disorders, recent studies have shed light on the regulation of this enzyme in arthropods, where new functions were discovered. This review will discuss the role of arthropod G6PDH in regulating redox homeostasis and immunometabolism and explore potential avenues for further research on this enzyme in various metabolic adaptations.

An Ixodes persulcatus Inhibitor of Plasmin and Thrombin Hinders Keratinocyte Migration, Blood Coagulation, and Endothelial Permeability.

The skin is the first host tissue that the tick mouthparts, tick saliva, and a tick-borne pathogen contact during feeding. Tick salivary glands have evolved a complex and sophisticated pharmacological arsenal, consisting of bioactive molecules, to assist blood feeding and pathogen transmission. In this work, persulcatin, a multifunctional molecule that targets keratinocyte function and hemostasis, was identified from Ixodes persulcatus female ticks. The recombinant persulcatin was expressed and purified and is a 25-kDa acidic protein with 2 Kunitz-type domains. Persulcatin is a classical tight-binding competitive inhibitor of proteases, targeting plasmin (Ki: 28 nM) and thrombin (Ki: 115 nM). It blocks plasmin generation on keratinocytes and inhibits their migration and matrix protein degradation; downregulates matrix metalloproteinase 2 and matrix metalloproteinase 9; and causes a delay in blood coagulation, endothelial cell activation, and thrombin-induced fibrinocoagulation. It interacts with exosite I of thrombin and reduces thrombin-induced endothelial cell permeability by inhibiting vascular endothelial-cadherin disruption. The multifaceted roles of persulcatin as an inhibitor and modulator within the plasminogen-plasmin system and thrombin not only unveil further insights into the intricate mechanisms governing wound healing but also provide a fresh perspective on the intricate interactions between ticks and their host organisms.

Molecular Detection of Rickettsia hoogstraalii in Hyalomma anatolicum and Haemaphysalis sulcata: Updated Knowledge on the Epidemiology of Tick-Borne Rickettsia hoogstraalii.

Ticks are hematophagous ectoparasites that transmit pathogens to animals and humans. Updated knowledge regarding the global epidemiology of tick-borne Rickettsia hoogstraalii is dispersed, and its molecular detection and genetic characterization are missing in Pakistan. The current study objectives were to molecularly detect and genetically characterize Rickettsia species, especially R. hoogstraalii, in hard ticks infesting livestock in Pakistan, and to provide updated knowledge regarding their global epidemiology. Ticks were collected from livestock, including goats, sheep, and cattle, in six districts of Khyber Pakhtunkhwa (KP) Pakistan. Overall, 183 hosts were examined, of which 134 (73.2%), including goats (number = 39/54, 72.2%), sheep (23/40, 57.5%), and cattle (71/89, 80%) were infested by 823 ticks. The most prevalent tick species was Rhipicephalus microplus (number = 283, 34.3%), followed by Hyalomma anatolicum (223, 27.0%), Rhipicephalus turanicus (122, 14.8%), Haemaphysalis sulcata (104, 12.6%), Haemaphysalis montgomeryi (66, 8.0%), and Haemaphysalis bispinosa (25, 3.03%). A subset of 210 ticks was selected and screened for Rickettsia spp. using PCR-based amplification and subsequent sequencing of rickettsial gltA and ompB fragments. The overall occurrence rate of R. hoogstraalii was 4.3% (number = 9/210). The DNA of Rickettsia was detected in Hy. anatolicum (3/35, 8.5%) and Ha. sulcata (6/49, 12.2%). However, no rickettsial DNA was detected in Rh. microplus (35), Rh. turanicus (35), Ha. montgomeryi (42), and Ha. bispinosa (14). The gltA and ompB fragments showed 99-100% identity with R. hoogstraalii and clustered phylogenetically with the corresponding species from Pakistan, Italy, Georgia, and China. R. hoogstraalii was genetically characterized for the first time in Pakistan and Hy. anatolicum globally. Further studies should be encouraged to determine the role of ticks in the maintenance and transmission of R. hoogstraalii in different hosts.

PEPCK and glucose metabolism homeostasis in arthropods.

The fat body is responsible for a variety of functions related to energy metabolism in arthropods, by controlling the processes of de novo glucose production (gluconeogenesis) and glycogen metabolism. The rate-limiting factor of gluconeogenesis is the enzyme phosphoenolpyruvate carboxykinase (PEPCK), generally considered to be the first committed step in this pathway. Although the study of PEPCK and gluconeogenesis has been for decades restricted to mammalian models, especially focusing on muscle and liver tissue, current research has demonstrated particularities about the regulation of this enzyme in arthropods, and described new functions. This review will focus on arthropod PEPCK, discuss different aspects to PEPCK regulation and function, its general role in the regulation of gluconeogenesis and other pathways. The text also presents our views on potentially important new directions for research involving this enzyme in a variety of metabolic adaptations (e.g. diapause), discussing enzyme isoforms, roles during arthropod embryogenesis, as well as involvement in vector-pathogen interactions, contributing to a better understanding of insect vectors of diseases and their control.

Universal Tick Vaccines: Candidates and Remaining Challenges.

Recent advancements in molecular biology, particularly regarding massively parallel sequencing technologies, have enabled scientists to gain more insight into the physiology of ticks. While there has been progress in identifying tick proteins and the pathways they are involved in, the specificities of tick-host interaction at the molecular level are not yet fully understood. Indeed, the development of effective commercial tick vaccines has been slower than expected. While omics studies have pointed to some potential vaccine immunogens, selecting suitable antigens for a multi-antigenic vaccine is very complex due to the participation of redundant molecules in biological pathways. The expansion of ticks and their pathogens into new territories and exposure to new hosts makes it necessary to evaluate vaccine efficacy in unusual and non-domestic host species. This situation makes ticks and tick-borne diseases an increasing threat to animal and human health globally, demanding an urgent availability of vaccines against multiple tick species and their pathogens. This review discusses the challenges and advancements in the search for universal tick vaccines, including promising new antigen candidates, and indicates future directions in this crucial research field.

Putative target sites in synganglion for novel ixodid tick control strategies.

Acaricide resistance is a global problem that has impacts worldwide. Tick populations with broad resistance to all commercially available acaricides have been reported. Since resistance selection in ticks and their role in pathogen transmission to animals and humans result in important economic and public health burden, it is essential to develop new strategies for their control (i.e., novel chemical compounds, vaccines, biological control). The synganglion is the tick central nervous system and it is responsible for synthesizing and releasing signaling molecules with different physiological functions. Synganglion proteins are the targets of the majority of available acaricides. In this review we provide an overview of the mode-of-action and resistance mechanisms against neurotoxic acaricides in ticks, as well as putative target sites in synganglion, as a supporting tool to identify new target proteins and to develop new strategies for tick control.

Low Genetic Polymorphism in the Immunogenic Sequences of Rhipicephalus microplus Clade C.

Rhipicephalus microplus tick highly affects the veterinary sector throughout the world. Different tick control methods have been adopted, and the identification of tick-derived highly immunogenic sequences for the development of an anti-tick vaccine has emerged as a successful alternate. This study aimed to characterize immunogenic sequences from R. microplus ticks prevalent in Pakistan. Ticks collected in the field were morphologically identified and subjected to DNA and RNA extraction. Ticks were molecularly identified based on the partial mitochondrial cytochrome C oxidase subunit (cox) sequence and screened for piroplasms (Theileria/Babesia spp.), Rickettsia spp., and Anaplasma spp. PCR-based pathogens-free R. microplus-derived cDNA was used for the amplification of full-length cysteine protease inhibitor (cystatin 2b), cathepsin L-like cysteine proteinase (cathepsin-L), glutathione S-transferase (GST), ferritin 1, 60S acidic ribosomal protein (P0), aquaporin 2, ATAQ, and R. microplus 05 antigen (Rm05Uy) coding sequences. The cox sequence revealed 100% identity with the nucleotide sequences of Pakistan's formerly reported R. microplus, and full-length immunogenic sequences revealed maximum identities to the most similar sequences reported from India, China, Cuba, USA, Brazil, Egypt, Mexico, Israel, and Uruguay. Low nonsynonymous polymorphisms were observed in ATAQ (1.5%), cathepsin-L (0.6%), and aquaporin 2 (0.4%) sequences compared to the homologous sequences from Mexico, India, and the USA, respectively. Based on the cox sequence, R. microplus was phylogenetically assembled in clade C, which includes R. microplus from Pakistan, Myanmar, Malaysia, Thailand, Bangladesh, and India. In the phylogenetic trees, the cystatin 2b, cathepsin-L, ferritin 1, and aquaporin 2 sequences were clustered with the most similar available sequences of R. microplus, P0 with R. microplus, R. sanguineus and R. haemaphysaloides, and GST, ATAQ, and Rm05Uy with R. microplus and R. annulatus. This is the first report on the molecular characterization of clade C R. microplus-derived immunogenic sequences.

Acaricides Resistance in Ticks: Selection, Diagnosis, Mechanisms, and Mitigation.

Ticks are blood-feeding ecto-parasites that have a cosmopolitan distribution in tropical and subtropical regions of the world. Ticks cause economic losses in the form of reduced blood, meat and dairy products, as well as pathogen transmission. Different acaricides such as organochlorines, organophosphates, formamidines (e.g. amitraz), synthetic pyrethroids, macrocyclic lactones, fipronil, and fluazuron are currently used sequentially or simultaneously to control tick infestations. Most acaricide treatments now face increasingly high chances of failure, due to the resistance selection in different tick populations against these drugs. Acaricide resistance in ticks can be developed in different ways, including amino acid substitutions that result in morphological changes in the acaricide target, metabolic detoxification, and reduced acaricide entry through the outer layer of the tick body. The current literature brings a plethora of information regarding the use of different acaricides for tick control, resistance selection, analysis of mutations in target sites, and resistance mitigation. Alternatives such as synergistic use of different acaricides, plant-derived phytochemicals, fungi as biological control agents, and anti-tick vaccines have been recommended to avoid and mitigate acaricide resistance. The purpose of this review was to summarize and discuss different acaricides applied for tick control, their mechanisms of action and resistance selection, genetic polymorphisms in their target molecules, as well as the approaches used for diagnosis and mitigation of acaricide resistance, specifically in Rhipicephalus microplus ticks.

Novel tick glutathione transferase inhibitors as promising acaricidal compounds.

Ticks are important ectoparasites with a worldwide distribution. The most commonly used method for tick control involves the use of acaricides. The main problem is that its indiscriminate use has led to the selection of resistant tick populations. Glutathione transferases (GSTs) are enzymes that play an important role in the detoxification of several types of compounds used in commercial tick control products. This work aims to find new bioactive molecules through in vitro assays with a panel of 160 molecules with putative inhibitory activity on the Rhipicephalus microplus GST enzyme (RmGST). Also, selected molecules were tested against GSTs from other tick species; Rhipicephalus decoloratus, Amblyomma variegatum, Rhipicephalus appendiculatus, and Haemaphysalis longicornis. The first screening on RmGST identified 30 compounds with the ability to modify the enzymatic activity of this enzyme. These compounds included different chemical families, like chalcones, diarylideneketones, flavone, thiazoles, thiourea, steroids, thiadiazines, indazoles, and hydrazine. The most potent compounds against RmGST belong to the diarylideneketones family with an inhibition concentration of 50% of activity (IC50) between 7-50 µM. Interestingly, one of the most potent compounds was also an inhibitor of the GST from other tick species. Experiments with R. microplus adults and larvae showed toxicity at 150 µM, suggesting a potential acaricidal effect of these molecules.

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.

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.

Molecular Survey and Spatial Distribution of Rickettsia spp. in Ticks Infesting Free-Ranging Wild Animals in Pakistan (2017-2021).

Rickettsia spp. associated with ticks infesting wild animals have been mostly neglected in several countries, including Pakistan. To address this knowledge gap, ticks were collected during 2017 to 2021 from wild animals including cats (Felis chaus), Indian hedgehogs (Paraechinus micropus), and wild boars (Sus scrofa). The collected ticks were morpho-molecularly identified and screened for the detection of Rickettsia spp. Morphologically identified ticks were categorized into four species of the genus Rhipicephalus: Rhipicephalus haemaphysaloides, Rh. turanicus, Rh. sanguineus sensu lato (s.l), and Rh. microplus. Among 53 wild animals examined, 31 were infested by 531 ticks, an overall prevalence of 58.4%. Adult female ticks were predominant (242 out of 513 ticks collected, corresponding to 46%) in comparison with males (172, 32%), nymphs (80, 15%) and larvae (37, 7%). The most prevalent tick species was Rh. turanicus (266, 50%), followed by Rh. microplus (123, 23%), Rh. sanguineus (106, 20%), and Rh. haemaphysaloides (36, 7%). Among the screened wild animals, wild boars were the most highly infested, with 268 ticks being collected from these animals (50.4%), followed by cats (145, 27.3%) and hedgehogs (118, 22.3%). Tick species Rh. haemaphysaloides, Rh. turanicus, and Rh. sanguineus were found on wild boars, Rh. haemaphysaloides, and Rh. microplus on cats, and Rh. turanicus on hedgehogs. In a phylogenetic analysis, mitochondrial cytochrome C oxidase 1 (cox1) sequences obtained from a subsample (120) of the collected ticks clustered with sequences from Bangladesh, China, India, Iran, Myanmar, and Pakistan, while 16S ribosomal DNA (16S rDNA) sequences clustered with sequences reported from Afghanistan, Egypt, India, Pakistan, Romania, Serbia, and Taiwan. Among Rickettsia infected ticks (10/120, 8.3%), Rh. turanicus (7/10, 70%), and Rh. haemaphysaloides (3/10, 30%) were found infesting wild boars in the districts Mardan and Charsadda. The obtained rickettsial gltA gene sequences showed 99% and ompA gene sequences showed 100% identity with Rickettsia massiliae, and the phylogenetic tree shows ompA clustered with the same species reported from France, Greece, Spain, and USA. This study emphasizes the need for effective surveillance and control programs in the region to prevent health risks due to tick-borne pathogens, and that healthy infested wild animals may play a role in the spread of these parasites.

Redox imbalance induces remodeling of glucose metabolism in Rhipicephalus microplus embryonic cell line.

Carbohydrate metabolism not only functions in supplying cellular energy but also has an important role in maintaining physiological homeostasis and in preventing oxidative damage caused by reactive oxygen species. Previously, we showed that arthropod embryonic cell lines have high tolerance to H2O2 exposure. Here, we describe that Rhipicephalus microplus tick embryonic cell line (BME26) employs an adaptive glucose metabolism mechanism that confers tolerance to hydrogen peroxide at concentrations too high for other organisms. This adaptive mechanism sustained by glucose metabolism remodeling promotes cell survival and redox balance in BME26 cell line after millimolar H2O2 exposure. The present work shows that this tick cell line could tolerate high H2O2 concentrations by initiating a carbohydrate-related adaptive response. We demonstrate that gluconeogenesis was induced as a compensation strategy that involved, among other molecules, the metabolic enzymes NADP-ICDH, G6PDH, and PEPCK. We also found that this phenomenon was coupled to glycogen accumulation and glucose uptake, supporting the pentose phosphate pathway to sustain NADPH production and leading to cell survival and proliferation. Our findings suggest that the described response is not atypical, being also observed in cancer cells, which highlights the importance of this model to all proliferative cells. We propose that these results will be useful in generating basic biological information to support the development of new strategies for disease treatment and parasite control.

The ecdysteroid receptor regulates salivary gland degeneration through apoptosis in Rhipicephalus haemaphysaloides.

BACKGROUND: It is well established that ecdysteroid hormones play an important role in arthropod development and reproduction, mediated by ecdysteroid receptors. Ticks are obligate hematophagous arthropods and vectors of pathogens. The salivary gland plays an essential role in tick growth and reproduction and in the transmission of pathogens to vertebrate hosts. During tick development, the salivary gland undergoes degeneration triggered by ecdysteroid hormones and activated by apoptosis. However, it is unknown how the ecdysteroid receptor and apoptosis regulate salivary gland degeneration. Here, we report the functional ecdysteroid receptor (a heterodimer of the ecdysone receptor [EcR] and ultraspiracle [USP]) isolated from the salivary gland of the tick Rhipicephalus haemaphysaloides and explore the molecular mechanism of ecdysteroid receptor regulation of salivary gland degeneration. METHODS: The full length of RhEcR and RhUSP open reading frames (ORFs) was obtained from the transcriptome. The RhEcR and RhUSP proteins were expressed in a bacterial heterologous system, Escherichia coli. Polyclonal antibodies were produced against synthetic peptides and were able to recognize recombinant and native proteins. Quantitative real-time PCR and western blot were used to detect the distribution of RhEcR, RhUSP, and RhCaspases in the R. haemaphysaloides organs. A proteomics approach was used to analyze the expression profiles of the ecdysteroid receptors, RhCaspases, and other proteins. To analyze the function of the ecdysteroid receptor, RNA interference (RNAi) was used to silence the genes in adult female ticks. Finally, the interaction of RhEcR and RhUSP was identified by heterologous co-expression assays in HEK293T cells. RESULTS: We identified the functional ecdysone receptor (RhEcR/RhUSP) of 20-hydroxyecdysone from the salivary gland of the tick R. haemaphysaloides. The RhEcR and RhUSP genes have three and two isoforms, respectively, and belong to a nuclear receptor family but with variable N-terminal A/B domains. The RhEcR gene silencing inhibited blood-feeding, blocked engorgement, and restrained salivary gland degeneration, showing the biological role of the RhEcR gene in ticks. In the ecdysteroid signaling pathway, RhEcR silencing inhibited salivary gland degeneration by suppressing caspase-dependent apoptosis. The heterologous expression in mammalian HEK293T cells showed that RhEcR1 interacts with RhUSP1 and induces caspase-dependent apoptosis. CONCLUSIONS: These data show that RhEcR has an essential role in tick physiology and represents a putative target for the control of ticks and tick-borne diseases.

Aedes fluviatilis cell lines as new tools to study metabolic and immune interactions in mosquito-Wolbachia symbiosis.

In the present work, we established two novel embryonic cell lines from the mosquito Aedes fluviatilis containing or not the naturally occurring symbiont bacteria Wolbachia, which were called wAflu1 and Aflu2, respectively. We also obtained wAflu1 without Wolbachia after tetracycline treatment, named wAflu1.tet. Morphofunctional characterization was performed to help elucidate the symbiont-host interaction in the context of energy metabolism regulation and molecular mechanisms of the immune responses involved. The presence of Wolbachia pipientis improves energy performance in A. fluviatilis cells; it affects the regulation of key energy sources such as lipids, proteins, and carbohydrates, making the distribution of actin more peripheral and with extensions that come into contact with neighboring cells. Additionally, innate immunity mechanisms were activated, showing that the wAflu1 and wAflu1.tet cells are responsive after the stimulus using Gram negative bacteria. Therefore, this work confirms the natural, mutually co-regulating symbiotic relationship between W. pipientis and A. fluviatilis, modulating the host metabolism and immune pathway activation. The results presented here add important resources to the current knowledge of Wolbachia-arthropod interactions.

Glucose metabolomic profile during embryogenesis in the tick Rhipicephalus microplus.

INTRODUCTION: Metabolomic approaches can assess the actual state of an organism's energy metabolism during a specific morphological event, providing a more accurate insight into the correlations between physiology and metabolic regulation. METHODS: The study of the metabolomic profile aim to identify the largest possible number of biomolecules in a certain organism or specific structures. For this purpose, mass spectrometry (MS) and chromatography have been used in the present study. OBJECTIVES: In this context, the aim of the present work is to evaluate the glucose metabolomic profile during embryogenesis in Rhipicephalus microplus tick, investigating the dynamics of nutrient utilization during tick embryo formation, as well as the control of glucose metabolism. RESULTS: We show that glycogen reserves are preferentially mobilized to sustain the energy-intensive process of embryogenesis. Subsequently, the increase in concentration of specific amino acids indicates that protein degradation would provide carbons to fuel gluconeogenesis, supplying the embryo with sufficient glucose and glycogen during development. CONCLUSION: Altogether, these results demonstrated the presence of a very refined catabolic and anabolic control during embryogenesis in R. microplus tick, suggesting the pronounced gluconeogenesis as a strategy to secure embryo development. Moreover, this research contributes to the understanding of the mechanisms that control glucose metabolism during tick embryogenesis and may aid the identification of putative targets for novel chemical or immunological control methods, which are essential to improve the prevention of tick infestations.

Interfering with cholesterol metabolism impairs tick embryo development and turns eggs susceptible to bacterial colonization.

Cholesterol is a known precursor of arthropod molecules such as the hormone 20-hydroxyecdysone and the antimicrobial boophiline, a component of tick egg wax coat. Because the cholesterol biosynthetic pathway is absent in ticks, it is necessarily obtained from the blood meal, in a still poorly understood process. In contrast, dietary cholesterol absorption is better studied in insects, and many proteins are involved in its metabolism, including Niemann-Pick C (NPC) transporter and acyl-CoA:cholesterol acyltransferase (ACAT), as well as enzymes to convert between free cholesterol and esterified cholesterol. The present work addresses the hypothesis that tick viability can be impaired by interfering with cholesterol metabolism, proposing this route as a target for novel tick control methods. Two drugs, ezetimibe (NPC inhibitor) and avasimibe (ACAT inhibitor) were added to calf blood and used to artificially feed Rhipicephalus microplus females. Results show that, after ingesting avasimibe, tick reproductive ability and egg development are impaired. Also, eggs laid by females fed with avasimibe did not hatch and were susceptible to Pseudomonas aeruginosa adhesion and biofilm formation in their surfaces. The immunoprotective potential of ACAT against ticks was also accessed using two selected ACAT peptides. Antibodies against these peptides were used to artificially feed female ticks, but no deleterious effects were observed. Taken together, data presented here support the hypothesis that enzymes and other proteins involved in cholesterol metabolism are suitable as targets for tick control methods.