Rhipicephalus microplus thyropin-like protein: Structural and immunologic analyzes.

Tick saliva has a pivotal function in parasitism. It has pharmacological and immunomodulatory properties, with several proteins reported in its composition. Thyroglobulin type-1 domain protease inhibitor (thyropin)-like proteins are found in tick saliva, but their function, properties and structures are poorly characterized. It has been reported that thyropins are capable of inhibiting cysteine peptidases present in antigen-presenting cells. To elucidate the role of thyropin-like proteins in ticks, we conducted in silico analysis and cloned an open reading frame from a thyropin-like protein found in Rhipicephalus microplus. The recombinant protein was successfully expressed, followed by immunological characterization and a vaccine trial against Rhipicephalus sanguineus in rabbits. Several differences are observed between thyropin-like proteins from hard and soft ticks, especially the number of thyroglobulin domains and predicted glycosylation pattern. Thyropin-like proteins also differ between postriata and metastriata ticks, the latter having a coil-domain at the C-terminal region and high number of predicted glycosylation sites. Overall, the data suggested divergence in thyropin-like proteins functions among ticks. The recombinant thyropin-like protein is immunogenic and the antibodies against it are able to recognize the native protein in tick saliva and tissues. While the recombinant protein does not elicit a protective response against R. sanguineus infestation, its characterization paves the way for further investigations aimed at determining the precise function of this protein in tick physiology.

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.

A longitudinal transcriptomic analysis of Rhipicephalus microplus midgut upon feeding.

Rhipicephalus microplus, a highly host-specific tick that primarily feeds on cattle, posing a significant threat to livestock production. The investigation of tick physiology is crucial for identifying potential targets in tick control. Of particular interest adult female ticks undergo a significant expansion of the midgut during feeding, leading to an over 100-fold increase in body weight. Beyond the functions of storing and digesting blood meals, the tick midgut plays a crucial role in acquiring and transmitting pathogens. However, our understanding of tick midgut physiology remains limited. In this study we conducted a comprehensive longitudinal transcriptome analysis of the midgut from adult female R. microplus ticks collected at various feeding stages, providing an overview of the transcriptional modulation in this organ as feeding progress. By employing a de novo assembly approach followed by coding-sequences (CDS) extraction, 60,599 potential CDS were identified. In preparation for functional annotation and differential expression analysis, transcripts that showed an average transcript per million (TPM) ≥ 3 in at least one of the biological conditions were extracted. This selection process resulted in a total of 10,994 CDS, which were categorized into 24 functional classes. Notably, our differential expression analysis revealed three main transcriptional profiles. In the first one, representing the slow-feeding stage, the most abundant functional classes were the "protein synthesis" and "secreted" groups, reflecting the highly active state of the tick midgut. The second profile partially accounts for the rapid-feeding stage, in which a high number of differentially expressed transcripts was observed. Lastly, the third transcriptional profile represents post-detached ticks. Notably the highest number of modulated transcripts was observed up to 48 h post-detachment (hpd), however no major differences was observed up to 168 hpd. Overall, the data presented here offers a temporal insight into tick midgut physiology, contributing to the identification of potential targets for the development of anti-tick control strategies.

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.

Rabbits as Animal Models for Anti-Tick Vaccine Development: A Global Scenario.

Studies evaluating candidate tick-derived proteins as anti-tick vaccines in natural hosts have been limited due to high costs. To overcome this problem, animal models are used in immunization tests. The aim of this article was to review the use of rabbits as an experimental model for the evaluation of tick-derived proteins as vaccines. A total of 57 tick proteins were tested for their immunogenic potential using rabbits as models for vaccination. The most commonly used rabbit breeds were New Zealand (73.8%), Japanese white (19%), Californians (4.8%) and Flemish lop-eared (2.4%) rabbits. Anti-tick vaccines efficacy resulted in up to 99.9%. Haemaphysalis longicornis (17.9%) and Ornithodoros moubata (12.8%) were the most common tick models in vaccination trials. Experiments with rabbits have revealed that some proteins (CoAQP, OeAQP, OeAQP1, Bm86, GST-Hl, 64TRP, serpins and voraxin) can induce immune responses against various tick species. In addition, in some cases it was possible to determine that the vaccine efficacy in rabbits was similar to that of experiments performed on natural hosts (e.g., Bm86, IrFER2, RmFER2, serpins and serine protease inhibitor). In conclusion, results showed that prior to performing anti-tick vaccination trials using natural hosts, rabbits can be used as suitable experimental models for these studies.

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.

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.

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.

Tick saliva-induced programmed death-1 and PD-ligand 1 and its related host immunosuppression.

The tick Rhipicephalus microplus is a harmful parasite of cattle that causes considerable economic losses to the cattle breeding industry. Although R. microplus saliva (Rm-saliva) contains several immunosuppressants, any association between Rm-saliva and the expression of immunoinhibitory molecules, such as programmed death (PD)-1 and PD-ligand 1 (PD-L1), has not been described. In this study, flow cytometric analyses revealed that Rm-saliva upregulated PD-1 expression in T cells and PD-L1 expression in CD14+ and CD11c+ cells in cattle. Additionally, Rm-saliva decreased CD69 expression in T cells and Th1 cytokine production from peripheral blood mononuclear cells. Furthermore, PD-L1 blockade increased IFN-γ production in the presence of Rm-saliva, suggesting that Rm-saliva suppresses Th1 responses via the PD-1/PD-L1 pathway. To reveal the upregulation mechanism of PD-1/PD-L1 by Rm-saliva, we analyzed the function of prostaglandin E2 (PGE2), which is known as an inducer of PD-L1 expression, in Rm-saliva. We found that Rm-saliva contained a high concentration of PGE2, and PGE2 treatment induced PD-L1 expression in CD14+ cells in vitro. Immunohistochemical analyses revealed that PGE2 and PD-L1 expression was upregulated in tick-attached skin in cattle. These data suggest that PGE2 in Rm-saliva has the potential to induce the expression of immunoinhibitory molecules in host immune cells.

Prediction, mapping and validation of tick glutathione S-transferase B-cell epitopes.

In search of ways to address the increasing incidence of global acaricide resistance, tick control through vaccination is regarded as a sustainable alternative approach. Recently, a novel cocktail antigen tick-vaccine was developed based on the recombinant glutathione S-transferase (rGST) anti-sera cross-reaction to glutathione S-transferases of Rhipicephalus appendiculatus (GST-Ra), Amblyomma variegatum (GST-Av), Haemaphysalis longicornis (GST-Hl), Rhipicephalus decoloratus (GST-Rd) and Rhipicephalus microplus (GST-Rm). Therefore, the current study aimed to predict the shared B-cell epitopes within the GST sequences of these tick species. Prediction of B-cell epitopes and proteasomal cleavage sites were performed using immunoinformatics algorithms. The conserved epitopes predicted within the sequences were mapped on the homodimers of the respective tick GSTs, and the corresponding peptides were independently used for rabbit immunization experiments. Based on the dot blot assay, the immunogenicity of the peptides and their potential to be recognized by corresponding rGST anti-sera raised by rabbit immunization in a previous work were investigated. This study revealed that the predicted conserved B-cell epitopes within the five tick GST sequences were localized on the surface of the respective GST homodimers. The epitopes of GST-Ra, GST-Rd, GST-Av, and GST-Hl were also shown to contain a seven residue-long peptide sequence with no proteasomal cleavage sites, whereas proteasomal digestion of GST-Rm was predicted to yield a 4-residue fragment. Given that a few proteasomal cleavage sites were found within the conserved epitope sequences of the four GSTs, the sequences could also contain a T-cell epitope. Finally, the peptide and rGST anti-sera reacted against the corresponding peptide, confirming their immunogenicity. These data support the claim that the rGSTs, used in the previous study, contain conserved B-cell epitopes, which elucidates why the rGST anti-sera cross-reacted to non-homologous tick GSTs. Taken together, the data suggest that the B-cell epitopes predicted in this study could be useful for constituting epitope-based GST tick vaccines.

Rhipicephalus microplus cystatin as a potential cross-protective tick vaccine against Rhipicephalus appendiculatus.

Rhipicephalus appendiculatus, the brown ear tick, is an important disease vector of livestock in eastern, central and southern Africa. Rhipicephalus appendiculatus acaricide resistance requires the search for alternative methods for its control. Cystatins constitute a superfamily of cysteine peptidase inhibitors vital for tick blood feeding and development. These inhibitors were proposed as antigens in anti-tick vaccines. In this work, we applied structural and biochemical approaches to characterize a new cystatin named R. appendiculatus cystatin 2a (Racys2a). Structural modeling showed that this new protein possesses characteristic type 2 cystatin motifs, besides conservation of other structural patterns along the protein. Peptidase inhibitory assays with recombinant Racys2a showed modulation of tick and host cathepsins involved in blood digestion and immune system responses, respectively. A heterologous tick challenge with R. appendiculatus in rabbits immunized with recombinant Rhipicephalus microplus cystatin 2c (rBmcys2c) was performed to determine cross-reactivity. Histological staining showed that rBmcys2c vaccination caused damage to the gut, salivary gland and ovary tissues in R. appendiculatus. Furthermore, cystatin vaccine reduced the number of fully engorged adult females in 11.5 %. Consequently, strategies to increase the protection rate are necessary, including the selection of two or more antigens to compose a vaccine cocktail.

Constituting a glutathione S-transferase-cocktail vaccine against tick infestation.

Cocktail vaccines are proposed as an attractive way to increase protection efficacy against specific tick species. Furthermore, such vaccines made with different tick antigens have the potential of cross-protecting against a broad range of tick species. However, there are still limitations to the selection of immunogen candidates. Acknowledging that glutathione S-transferases (GSTs) have been exploited as vaccines against ticks and other parasites, this study aimed to analyze a GST-cocktail vaccine as a potential broad-spectrum tick vaccine. To constitute the GST-cocktail vaccine, five tick species of economic importance for livestock industry were studied (Rhipicephalus appendiculatus, Rhipicephalus decoloratus, Rhipicephalus microplus, Amblyomma variegatum, and Haemaphysalis longicornis). Tick GST ORF sequences were cloned, and the recombinant GSTs were produced in Escherichia coli. rGSTs were purified and inoculated into rabbits, and the immunological response was characterized. The humoral response against rGST-Rd and rGST-Av showed a stronger cross-reactivity against heterologous rGSTs compared to rGST-Hl, rGST-Ra, and rGST-Rm. Therefore, rGST-Rd and rGST-Av were selected for constituting an experimental rGST-cocktail vaccine. Vaccination experiment in rabbits showed that rGST-cocktail caused 35% reduction in female numbers in a Rhipicephalus sanguineus infestation. This study brings forward an approach to selecting immunogens for cocktail vaccines, and the results highlight rGST-Rd and rGST-Av as potentially useful tools for the development of a broad-spectrum tick vaccine.

Expression profile of Rhipicephalus microplus vitellogenin receptor during oogenesis.

The vitellogenin receptor (VgR), which belongs to the low-density lipoprotein receptors (LDLR) family, regulates the absorption of yolk protein accumulated in developing oocytes during oogenesis. In the present study, the full sequence of Rhipicephalus microplus VgR (RmVgR) and the partial sequence of Rhipicephalus appendiculatus VgR (RaVgR) ORF were determined and cloned. The RmVgR amino acid sequence contains the five highly conserved structural motifs characteristic of LDLR superfamily members, the same overall structure as observed in other species. Phylogenetic analysis separated VgRs in two major groups, corresponding to receptors from acarines and insects. Consistent with observations from other arthropods, RmVgR was specifically expressed in the ovarian tissue and its peak of expression occurs in females that are detaching from the host. Silencing with RmVgR dsRNA reduced VgR expression, which resulted in reduced fertility, evidenced by a decrease in the number of larvae. The present study confirms RmVgR is a specific receptor involved in yolk protein uptake and oocyte maturation in R. microplus, playing an important role in tick reproduction.

A Coxiella mutualist symbiont is essential to the development of Rhipicephalus microplus.

The cattle tick Rhipicephalus microplus is a hematophagous ectoparasite that causes important economic losses in livestock. Different species of ticks harbor a symbiont bacterium of the genus Coxiella. It was showed that a Coxiella endosymbiont from R. microplus (CERM) is a vertically transmitted mutualist symbiont, comprising 98% of the 16S rRNA sequences in both eggs and larvae. Sequencing of the bacterial genome revealed genes for biosynthetic pathways for several vitamins and key metabolic cofactors that may provide a nutritional complement to the tick host. The CERM was abundant in ovary and Malpighian tubule of fully engorged female. Tetracycline treatment of either the tick or the vertebrate host reduced levels of bacteria in progeny in 74% for eggs and 90% for larvae without major impact neither on the reproductive fitness of the adult female or on embryo development. However, CERM proved to be essential for the tick to reach the adult life stage, as under antibiotic treatment no tick was able to progress beyond the metanymph stage. Data presented here suggest that interference in the symbiotic CERM-R. microplus relationship may be useful to the development of alternative control methods, highlighting the interdependence between ticks and their endosymbionts.

Effect of recombinant glutathione S-transferase as vaccine antigen against Rhipicephalus appendiculatus and Rhipicephalus sanguineus infestation.

The ticks Rhipicephalus appendiculatus and Rhipicephalus sanguineus are the main vectors of Theileria parva and Babesia spp. in cattle and dogs, respectively. Due to their impact in veterinary care and industry, improved methods against R. appendiculatus and R. sanguineus parasitism are under development, including vaccines. We have previously demonstrated the induction of a cross-protective humoral response against Rhipicephalus microplus following vaccination with recombinant glutathione S-transferase from Haemaphysalis longicornis tick (rGST-Hl), suggesting that this protein could control tick infestations. In the present work, we investigated the effect of rGST-Hl vaccine against R. appendiculatus and R. sanguineus infestation in rabbits. In silico analysis revealed that GST from H. longicornis, R. appendiculatus and R. sanguineus have >80% protein sequence similarity, and multiple conserved antigenic sites. After the second vaccine dose, rGST-Hl-immunized rabbits showed elevated antibody levels which persisted until the end of experiment (75 and 60 days for R. appendiculatus and R. sanguineus, respectively). Western blot assays demonstrated cross-reactivity between anti-rGST-Hl antibodies and native R. appendiculatus and R. sanguineus GST extracts from ticks at different life stages. Vaccination with rGST-Hl decreased the number, weight, and fertility of engorged R. appendiculatus adults, leading to an overall vaccine efficacy of 67%. Interestingly, histological analysis of organ morphology showed damage to salivary glands and ovaries of R. appendiculatus adult females fed on vaccinated animals. In contrast, rGST-Hl vaccination did not affect R. appendiculatus nymphs, and it was ineffective against R. sanguineus across the stages of nymph and adult. Taken together, our results show the potential application of rGST-Hl as an antigen in anti-tick vaccine development, however indicating a broad difference in efficacy among tick species.

Molecular and functional characterization of Bm05br antigen from Rhipicephalus microplus.

Rhipicephalus microplus is a cattle-specific tick, causing considerable losses in the livestock industry. The identification of molecules responsible for modulation of host defenses during different parasite stages can help in the development of alternative methods, such as vaccination, to control tick infestations. Hq05, a protein of unknown function identified in the tick Haemaphysalis qinghaiensis, induced a significant protective immune response when used as a vaccine in sheep. In the present study, we investigated Bm05br, the Hq05 homologous gene from R. microplus. Besides H. qinghaiensis, Bm05br homologous found in other tick species such as Rhipicephalus annulatus, Rhipicephalus sanguineus sensu lato, Haemaphysalis longicornis and Ixodes scapularis were comparatively analyzed. Bm05br expression profile in different R. microplus tissues and life-stages was determined by qRT-PCR and Western blot. Bm05br was detected in ovaries, salivary glands and the fat body of both partially and fully engorged females. The highest transcription levels were observed in partially engorged females fat body and salivary glands. Gene knockdown by RNAi reduced egg hatching rate and the weight of tick larvae obtained from treated group, when compared to controls. These results indicate that Bm05br may be involved in R. microplus reproduction. Together with its distribution and high sequence conservation across different tick species, our data suggest Bm05br as a potential antigen for development of a multispecies anti-tick vaccine.

A revision of two distinct species of Rhipicephalus: R. microplus and R. australis / Uma revisão sobre duas espécies distintas de Rhipicephalus: R. microplus e R. Australis

ABSTRACT: Rhipicephalus ( Boophilus) species are monoxenous ticks with seasonal distribution in tropical and subtropical regions. For many years, Rhipicephalus micropluswas considered as a single species; however, further analysis split these ticks into two distinct species. Because R. microplusand R. australisshare similar attributes, it is hard to discriminate these two species and explain the changes in the classification of these parasites over the past decades. The reappearance of R. australisis an outcome of new research, which has afforded to better characterize these probably cryptic species. Evidence based on morphological features, the lack of conspecificity, microsatellite markers, mitochondrial 12S and 16S ribosomal DNA, and mitochondrial genome supports the re-classification of R. microplusas different species. Therefore, populations of R. microplusfrom Australia, Cambodia, Philippines, Indonesia, New Caledonia, Borneo, New Guinea, Tahiti and parts of Southeast Asia were recently reinstated as R. australis. Moreover, a better knowledge on the speciation between these two species could pave the way to important advances in tick control strategies.

RESUMO: As espécies pertencentes ao gênero Rhipicephalus (Boophilus) são carrapatos monoxenos de distribuição sazonal em regiões tropicais e subtropicais. Por muito anos, duas espécies de carrapatos foram consideradas como Rhipicephalus microplus.Contudo, estudos recentes reclassificaram esse carrapato em duas espécies: R. micropluse R. australis. Em razão de diversas semelhanças entre R. micropluse R. australis,distinguir essas duas espécies torna-se uma tarefa árdua, o que explica as mudanças de classificação dessas espécies nas últimas décadas. O reaparecimento da espécie R. australissurge com novas pesquisas, resultado de uma melhor caracterização dessas prováveis espécies crípticas. Evidências baseadas em análises das características morfológicas, na ausência de co-especificidade, em marcadores de microssatélites, no DNA ribossomal mitocondrial 12S e 16S, assim como no genoma mitocondrial, suportam a re-classificação de R. micropluscomo duas espécies distintas. Nesse sentido, populações de R. microplusda Austrália, Camboja, Nova Caledônia, Bornéo, Filipinas, Nova Guiné, Indonésia e Taiti foram recentemente renomeadas como R. australis. Além disso, um melhor entendimento sobre a especiação e localização dessas duas espécies pode trazer avanços importantes para melhorar as estratégias de controle desses carrapatos.

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.

Rhipicephalus microplus and Ixodes ovatus cystatins in tick blood digestion and evasion of host immune response.

BACKGROUND: Cystatins are a group of cysteine protease inhibitors responsible for physiological proteolysis regulation and present in a wide range of organisms. Studies about this class of inhibitors in parasites have contributed to clarify their roles in important physiological processes, like blood digestion and modulation of host immune response during blood feeding. Thus, cystatins are a subject of research on the development of new parasite control methods. Additionally, the characterization of proteins shared by different parasite species represents a valuable strategy to find potential targets in multi-species control methods. However, cystatin functions in ticks remain undetermined, especially in Rhipicephalus microplus and Ixodes ovatus, two species that affect livestock and human health, respectively. METHODS: Here we report the inhibitory profile of two R. microplus (BrBmcys2b and BrBmcys2c) and one I. ovatus (JpIocys2a) cystatins to commercial cathepsins B, C, and L. The presence of native cystatins in R. microplus tissues was analyzed using sera against recombinant BrBmcys2b and BrBmcys2c. Also, a peptide from JpIocys2a was synthesized for rabbit immunization, and this serum was used to analyze the cross antigenicity between R. microplus and I. ovatus cystatins. RESULTS: Enzymatic inhibition profile of tick cystatins shows a distinct modulation for cathepsins related to tick blood digestion and evasion of host immune response. Furthermore, BrBmcys2b was detected in saliva and different tissues along tick stages, while BrBmcys2c was detected mainly in gut from partially engorged R. microplus females, demonstrating a distinct pattern of cystatin expression, secretion and traffic between tick tissues. Moreover, phylogenetic analysis suggests that JpIocys2a belongs to the group of tick gut secreted cystatins. Finally, cross-antigenicity assays revealed that antibodies against the JpIocys2a peptide recognize native and recombinant R. microplus cystatins. CONCLUSION: The presence of these proteins in different tissues and their ability to differentially inhibit cathepsins suggest distinct roles for JpIocys2a, BrBmcys2b, and BrBmcys2c in blood digestion, egg and larvae development, and modulation of host immune response in tick physiology. The cross-antigenicity between native and recombinant cystatins supports further experiments using JpIocys2a, BrBmcys2b, and BrBmcys2c as vaccine antigens.