Abstract
Control of complex parasites via vaccination remains challenging, with the current combination of vaccines and small drugs remaining the choice for an integrated control strategy. Studies conducted to date, are providing evidence that multicomponent vaccines will be needed for the development of protective vaccines against endo- and ectoparasites, though multicomponent vaccines require an in-depth understanding of parasite biology which remains insufficient for ticks. With the rapid development and spread of acaricide resistance in ticks, new targets for acaricide development also remains to be identified, along with novel targets that can be exploited for the design of lead compounds. In this study, we analysed the differential gene expression of Rhipicephalus microplus ticks that were fed on cattle vaccinated with a multi-component vaccine (Bm86 and 3 putative Bm86-binding proteins). The data was scrutinised for the identification of vaccine targets, small drug targets and novel pathways that can be evaluated in future studies. Limitations associated with targeting novel proteins for vaccine and/or drug design is also discussed and placed into the context of challenges arising when targeting large protein families and intracellular localised proteins. Lastly, this study provide insight into how Bm86-based vaccines may reduce successful uptake and digestion of the bloodmeal and overall tick fecundity.
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Introduction
The Asiatic blue tick, Rhipicephalus microplus (Canestrini), is considered one of the most successful tick species of cattle worldwide due to its adaptability to various climatic conditions (Nyangiwe et al. 2017), its displacement of native tick species (Coetzer and Tustin 2004; Tønnesen et al. 2004; Muhanguzi et al. 2020; Nyangiwe et al. 2017), rapid development of acaricide resistance (Lovis et al. 2013; Nyangiwe et al. 2013; Rodriguez-Vivas et al. 2018), and its ability to transmit a variety of economically important tick-borne diseases (Pereira et al. 2022). Currently, acaricides form the cornerstone of tick control globally, but with the growing evidence of resistance against all major classes of acaricides (Dzemo et al. 2022), there is an urgent need for complimentary and/or alternative tick control strategies.
Currently high-throughput data is being mined for novel anti-parasitic drug targets, and recent examples include evaluation of: piperazine derivatives against Leishmania species (Schadich et al. 2022), acetylcholinesterase inhibitors and targets for R. microplus (Cerqueira et al. 2022), as well as transmission blocking drugs against an array of tick-borne diseases (Schorderet-Weber et al. 2018). As more genomic and transcriptomic data for parasites and pathogens of interest become available, the focus has shifted from classical biochemical screening of chemical libraries (Woods and Williams 2007) to in silico screening of targets including: analysis of the physiochemical properties of the protein, structural modelling, high-throughput screening of large compound libraries and drug docking (e.g., Shigella dysenteriae) (Jalal et al. 2022). For ticks, however, the lack of protein crystal structures remains a bottleneck, along with the vast amount of uncharacterized tick-specific proteins, hinder high-quality protein modelling. The next challenge remains the bioavailability and delivery of the antiparasitic drug to the correct protein target (Lipinski et al. 1997), as well as the druggability of these targets (Kozakov et al. 2015; Cimermancic et al. 2016; Fauman et al. 2011).
In the pursuit of novel tick control, tick vaccines have been explored as a complementary approach to chemical control (Stutzer et al. 2018). The rationale for the identification and selection of target antigens remains a severe bottleneck in vaccine development. Early conventional methods, such as protein fractionation, followed by immunization and challenge in animal models, have been adapted to more modern ‘genomics to vaccinology’ approaches (i.e., reverse vaccinology). As the number of genomes being sequenced increases, data on associated transcriptomes and encoded proteomes also increases, and therefore data on various tissues over the duration of the parasite lifecycle could be obtained (Maritz-Olivier et al. 2012; Garcia et al. 2020; Tirloni et al. 2020; Stutzer et al. 2013). This provides valuable insight into the kinetics of antigen expression which is a crucial parameter when selecting antigens for vaccines (Moxon et al. 2019). In silico analyses can identify open reading frames used (among others) to predict similarity to other known vaccine antigens, as well as determine protein characteristics such as putative subcellular localization (e.g., surface expression, secretion, etc.) (Moxon et al. 2019). This can be followed by cloning and recombinant protein production of promising antigens, for vaccination and challenge studies, as well as raising specific antibodies for cell culture studies (e.g. expression library immunization or ELI) (Almazán et al. 2003). As the tools and criteria for in silico identification of surface proteins and protective antigens improve, it is continuously shaping the future of rational vaccine design.
Despite the strides in identification of new vaccine candidate antigens, Bm86-based vaccines remain the most effective against R. microplus infestations under field conditions to date (Ndawula and Tabor 2020; Pereira et al. 2022). The Bm86 antigen provided the first proof of concept for a purified single antigen tick vaccine (De La Fuente et al. 2007), and currently the Bm86-based vaccines Gavac™ (and Gavac Plus™) (Herber-Biotec S.A., CIGB, Camagüey, Cuba), as well as Bovimune Ixovac (Lapisa, Michoacan, Mexico) (Blecha et al. 2018), are available in Latin America. A better and deeper understanding of host–parasite interactions (e.g. detailed host immune response analyses), as well as parasite biology (i.e., the metabolic pathways and proteins involved, throughout the life cycle), is desperately needed. For R. microplus, more than 38,827 putative gene loci encoding around 24,785 putative proteins are contained in the most recent genome on the NCBI database (GCA_013339725.1), and more than half of these proteins are unique to the tick and remain largely unannotated (Barrero et al. 2017). With such a large and repetitive genome and probable variability in transcriptome kinetics (influenced by life stage and environmental factors), it can be hypothesized that multi-stage/multi-component cocktail vaccines (i.e., including more than one antigen) could enhance the level of protection afforded against infestation (Pereira et al. 2022). This appears to be a trend for vaccines against other complex parasites/pathogens, in example vaccines against: helminths (Maizels 2021), endoparasites such as malaria (Pirahmadi et al. 2021), Theileria (Atchou et al. 2020; Saaid et al. 2020), Babesia (Rathinasamy et al. 2019) and intracellular bacteria (Osterloh 2022), as well as ticks (Pereira et al. 2022; Costa et al. 2021). However, some cocktail vaccines tested against R. microplus did not live up to expectations (Pereira et al. 2022), and this is likely due to: antigen type (i.e., full-length, peptide, chimera, etc.) and quality; protein production platform (e.g., bacterial, yeast, etc.); antigen concentration; antigen-adjuvant interaction; antigenic competition; or even animal genetics, where information on the impact on the host immune system is incomplete (Ndawula and Tabor 2020; Stutzer et al. 2018).
In this study, we aimed to characterize the compensatory mechanisms used by R. microplus ticks to overcome the deleterious effects of host immunity when feeding on cattle hosts vaccinated with a Bm86-based multicomponent formulation (i.e., containing Bm86 and three putative Bm86-binding proteins, University of Pretoria invention disclosure) using DNA microarrays and subsequent differential gene expression profiling. We wanted to determine whether high-throughput transcriptomics can be used to elucidate biological reasons for why a vaccine can fail to confer sufficient protection (e.g. compensatory mechanisms), as well as whether this data can be used for the rational selection of additional vaccine candidates based on their putative function and accessibility to the host immune system (i.e., extracellular membrane bound or secreted). The data generated in this study was also evaluated, based on published examples, for identification of novel pathways and druggable targets to direct future efforts in next generation chemical control.
Material and methods
Rhipicephalus microplus tick strain
Pathogen free Rhipicephalus microplus larvae, from a laboratory bred strain of South African origin, were obtained from ClinVet International (Bloemfontein, South Africa). Shortly, batches of 8 to 10 engorged females fed on donor cattle are placed into 200 ml conical flasks and allowed to lay eggs under controlled conditions (28 °C, 80% humidity). The resultant larvae are used for infestation trials within 2 months of occlusion.
Cattle vaccination trial
6- to 8-month-old Holstein–Friesian (Bos taurus) female calves were housed at the Onderstepoort Veterinary Animal Research Unit (OVARU), University of Pretoria, Onderstepoort, South Africa. Calves (6–7 months of age) were treated prophylactically, a week ahead of commencement of vaccination studies, with Engemycin 10% 20 mg/kg intramuscularly, Baycox 5% 3 ml/10 kg per os, Valbazen 1 ml/10 kg per os, Berenil 3.5 mg/kg subcutaneously and Kyroligo 5 ml/calve intramuscularly, as well as dipped with Amitraz (Coopers Triatix 12.5%, MSD Animal Health, South Africa), as per manufacturer’s instructions, to remove any ectoparasites prior to commencement of vaccination. The health of the calves was further evaluated by examining peripheral blood smears for haemoparasite screening and haematocrit determination from drawn blood, as well as monitoring calf weights (weekly) and temperatures (daily) for the duration of the study. Ethical clearance was granted for the acquisition and rearing of ticks, as well as to study the effect of vaccination by the University of Pretoria Animal Ethics Committee (Project numbers: EC036-13) and from the Department of Agriculture, Land Reform and Rural Development (Section 20 of the Animal Diseases Act 1984, reference number: 12/11/1/8/1). Cattle were divided into two groups of four individuals (n = 4) at random, i.e., a adjuvant/saline control group and a test group vaccinated with Bm86 in combination with three novel R. microplus antigens (i.e., RmAg1, RmAg2 and RmAg3) at 50 µg each. Antigens were formulated with 1:1 (v/v) Montanide ISA 71 VG (Seppic, France) and a final volume of 1 ml was administered subcutaneously in the neck. Recombinant Bm86 was derived from the Mexican susceptible CENAPA strain, Genbank accession number ACR19243, produced in Pichia pastoris and kindly supplied by Prof J de la Fuente, Spain. The putative binding proteins (1–3) were produced in Escherichia coli (Biologics Corp, USA). A first booster immunisation was administered 4 weeks after the initial immunisation (i.e., Day 28), and a second booster after an additional 14 days (i.e., Day 42). An estimated 4000 larvae were used per animal for full body infestation 10 days after administration of the second booster. Ticks were allowed to complete their life cycle and feed to engorgement. Dropped/fully engorged female ticks were collected, counted, weighed, and placed in small, aerated plastic containers and incubated at 28 °C (80% humidity) in a humidifying incubator for ovipositing. The weight of laid eggs per female was measured.
Tick vaccine efficacy (E) was calculated as \(E \left( \% \right) = 100\left( {1 - \mathop \prod \nolimits_{k = 1}^{n} a_{k} } \right)\) where \(\mathop \prod \nolimits_{k = 1}^{n} a_{k}\) represents the reduction in the studied developmental processes \((k)\) in ticks fed on vaccinated cattle as compared to the control fed on adjuvant/saline injected cattle (Cunha et al. 2013). The efficacy of the vaccine was calculated considering the effect on the reduction of tick infestations (i.e., log-transformed number of dropped female ticks per animal), feeding efficiency (i.e., the weight of dropped female ticks) and oviposition (i.e., the weight of eggs laid per dropped female tick) as 100 \(\left[ {1 - \left( {{\text{CRT }} \times {\text{ CRW }} \times {\text{ CRO}}} \right)} \right]\) where CRT, CRW and CRO are the reduction in the number of female ticks, weight of female ticks and oviposition compared to the control group, respectively.
Data were analysed statistically by a one-tailed Student’s t test using a significance level of 0.05. Before determining significance, the Bartlett’s test for homogeneity of variances was performed on all data to test that variance are equal between the control and test groups. Using these results, either a one-tailed Student’s t test with equal- or unequal variance was performed. To determine statistical outliers, the interquartile range (IQR) of the number of ticks per group, as well as the first and third quartiles were calculated using Microsoft Excel 2010. The IQR was multiplied by 1.5 and subtracted from the first quartile and added to the third quartile. Data values that fall out of this range were considered statistical outliers.
Enzyme-linked immunosorbent assay
Blood samples were collected from cattle before commencement of the study (i.e., Day 0), 13 days after the first booster (i.e., Day 42) and 14 days after the second booster (i.e., Day 56). Briefly, 96 well MicroWell™ MaxiSorp™ flat bottom plates (Nunc, Denmark) were coated with 100 ng antigen diluted in TBS (Tris-buffered saline, 25 mM Tris–HCl, 150 mM NaCl, pH 7.5). Plates were dried overnight at room temperature, followed by four washing steps with 350 µl TBS-T (TBS containing 0.05% v/v Tween 20, pH 7.5). Blocking was performed using 350 µl blocking buffer (TBS-T containing 0.5% w/v casein, pH 7.5) for one hour at room temperature, followed by four washing steps. Serum samples were diluted in blocking buffer, added to the plates, incubated for 1 h at room temperature and washed four times. A 1:100 (for RmAg1, RmAg2 and RmAg3) and a 1:4000 (for Bm86) dilutions were prepared from primary serum collected on day 0, 42 and 56. Total bound immunoglobulin antibodies were detected by incubation with 1:6000 (v/v) diluted horseradish peroxidase-conjugated goat α-bovine IgG (Abcam Biotechnology company, UK). Following a final wash cycle, 200 µl substrate solution (2.2 mM o-phenylenediamine dihydrochloride, 0.05 M phosphate-citrate buffer, pH 5, 0.012% fresh H2O2) were added and the reaction monitored using the Multiskan Plus reader (Thermo Fisher Scientific, USA) at 450 nm with a reference filter at 690 nm. Antibody titres in vaccinated cattle were expressed as the OD450nm (ODcattle sera − ODbaseline) and compared between the different time points by one-way ANOVA (α = 0.05).
Isolation of total RNA and cDNA synthesis
Twenty semi-engorged female ticks from each of the four cattle allocated in the combination vaccinated and control (untreated) groups (i.e., 80 ticks per group), respectively, were collected 20 days post infestation. Midgut tissues were collected by dissection and homogenized in TRI Reagent (Sigma-Aldrich, Germany), frozen in liquid nitrogen and stored at −80 °C until RNA isolation was performed. Total RNA isolation, first strand cDNA synthesis, RNA hydrolysis and cDNA purifications were performed as per the methods outlined by Stutzer et al. (2013).
DNA microarrays
Microarray analysis was performed using a custom 4 × 44 K microarray slide platform designed from the transcriptome data (RmiGI Version 3 Gene Index) derived from the genome assembly of R. microplus (Wikel strain). A total of 28 913 sequences were submitted online for array design using the Agilent 4 × 44 k microarray and eArray microarray design platforms (https://earray.chem.agilent.com/earray/). Vector sequences derived from EST data were removed. Using the standard base composition probe design strategy, 60 mer probes representing the full complement of transcripts were designed. As controls, duplicates of half of the transcripts were distributed at random in the array design together with other standard incorporated control probes. A reference pool experimental design was used for transcriptome analysis of R. microplus fed on control and vaccinated (i.e., Bm86 and putative binding proteins combinatorial) cattle. Dye coupling via incorporated amoinoallyl dUTPs was achieved by the addition of 2.5 µl of Cyanine 3-dCTP (reference pool) or Cyanine 5-dCTP (test sample) (Amersham Biosciences, UK). Equivalent concentrations of the dye coupled sample and reference pool cDNA (50 pmol each) were hybridised to the microarray slide for 17 h at 65 °C using the Agilent Gene Expression Hybridisation Kit (Agilent Technologies, USA) according to the manufacturer’s recommendations. Following hybridisation, the microarray slides were washed, rinsed, and dried prior to scanning using the Axon GenePix™ 4000B microarray laser scanner (Molecular Devices, USA).
In silico data analyses and functional annotation of differentially expressed transcripts
DNA microarray data analysis was performed as per the method outlined by Stutzer et al. (2013), using the Robust spline method for within-array normalisation (M values). The threshold for significance was based on adjusted P < 0.01 and the upper and lower 1% log2FC distribution of differentially expressed transcripts in the vaccinated group relative to the control group. Normalized microarray data was submitted to the GEO repository of the National Centre for Biotechnology Information on accession no GSE221646.
Microarray probe sequences were annotated using the comprehensive annotation suite Blast2GO (http://www.blast2go.com) (Conesa et al. 2005). Additional similarity searches were performed using BLASTX or BLASTP searches against selected databases, i.e., the tick annotation release 100, based on the assembly ASM1333972v1 (GCF_013339725.1) (Jia et al. 2020), the Rmi2.0 annotation based on assembly GCA_002176555.1 (Barrero et al. 2017) and the RefSeq Invertebrate Protein database with sequences containing Acari [organism] protein sequences obtained from GenBank (Mistry et al. 2020).
Semi-quantitative real-rime PCR validation of array results
To verify the differential gene expression results obtained from DNA microarray analysis, RT-qPCR analysis was performed. Gene specific primers were designed for 10 transcripts that were up- and downregulated, respectively, for vaccinated vs. control group (Supplementary Table 1). Semi-quantitative PCR was performed as per the method outlined by Stutzer et al. (2013), using 10 pmol of oligonucleotide primer pairs corresponding to the selected sequences that were differentially expressed. The mRNA levels were normalised against two reference genes namely, elongation factor I-alpha (ELF1α) and ribosomal protein L4 (RLP4), that were previously validated to have stable expression in R. microplus (Nijhof et al. 2009). All reactions were carried out in duplicate. The Quant Studio 12 K-flex system was used for all reactions with its corresponding software (Life Technologies Biotechcompany, USA). Quantitative analysis was performed using qBasePLUS software (Biogazelle, Zwijnaarde, Belgium–http://www.qbaseplus.com) (Hellemans et al. 2007). The relative transcript levels for selected genes were evaluated using the extracted calibrated normalised relative quantities (CNRQ) and expressed as a fold change (on a log2 scale) relative to the reference genes selected. These normalised values were used to calculate the fold change for selected transcripts. Analysis of variance (ANOVA) was used to determine significant differential gene expression compared between the test and control groups (α = 0.05).
Results and discussion
Bovine antibody response to vaccination
Indirect ELISA-results demonstrated that no antigen-specific IgG response and cross-reactive responses against Bm86 were observed in control cattle. Cattle from the test group developed an antibody response against Bm86 (Fig. 1A) which increased significantly after the second vaccination (P = 1.04E−04) and the third vaccination (P = 0.023), before cattle were challenged with larvae on day 55. Weak immune responses were observed against RmAg1 (Fig. 1B) and RmAg2 (Fig. 1C), while a significant response against RmAg3 (P = 3.27E−04) was achieved (Fig. 1D).
Effect of vaccination on R hipicephalus microplus feeding
A 44.7% reduction in the number of engorged female ticks from cattle vaccinated with the combinatorial Bm86 vaccine, compared to the control group, was observed (P = 0.033; Table 1). Though the tick- and egg weights were not significantly affected, the calculated protective efficacy of the vaccine was 40.3% (Table 1).
Microarray data validation by RT-qPCR
To validate the microarray data, relative RT-qPCR was performed for ten most upregulated and ten most downregulated transcripts. Analysis of variance (ANOVA) indicated that the expression levels of all 20 transcripts were significantly differentially expressed in the vaccinated group compared to the control group (P < 0.05). An overall concordance was observed in the direction and magnitude of fold change values obtained from microarray and RT-qPCR analyses, thereby independently validating the results obtained from the microarray experiment (Fig. 2).
Functional annotations of differentially expressed transcripts
Blast2GO annotation showed 54% of the differentially expressed transcripts having a significant BLAST hit against the tick annotation release 100, based on the assembly ASM1333972v1 (GCF_013339725.1) (Jia et al. 2020). Some 91% and 83% of the differentially expressed transcripts with significant BLAST hits were assigned GO and InterProScan annotations, respectively.
Differentially expressed transcripts involved in digestion of the bloodmeal and vitellogenesis
In ticks, digestion of albumin and haemoglobin from the bloodmeal takes place in the acidic intracellular compartments of the gut epithelium. In the case of haemoglobin, the protein is processed into large fragments by endopeptidases namely: cathepsin D supported by cathepsin L and legumain (Cruz et al. 2010; Fogaça et al. 1999; Sojka et al. 2013). Subsequent proteolysis is mediated by the action of exopeptidases through the dipeptidase activities of cathepsins B and C. It is proposed that monopeptidases, including serine carboxypeptidase and leucine aminopeptidase, participate in the liberation of free amino acids (Cruz et al. 2010; Fogaça et al. 1999; Sojka et al. 2013). As vaccination with Bm86 is known to disrupt digestion and leakage of haemoglobin into the body cavity of ticks, the so called “red phenotype” (Agbede and Kemp 1986), we anticipated the differential expression of numerous proteases and protease inhibitors in ticks fed on vaccinated animals.
Three putative cathepsin D-like transcripts were downregulated in ticks feeding on the vaccinated cattle (T0010166, TC16620 and CV445464), along with a putative legumain-like transcript (TC17290). This highlights disruption of the first steps of digesting haemoglobin into large fragments. In contrast, two putative cathepsin L encoding transcripts (TC15655 and Contig1628) were significantly upregulated (Table 2). Transcript TC15655 has 98% sequence identity to BmCL1 which has been shown to localize to the inside of vesicles within the midgut epithelium of partially engorged females where is it proposed to play a role in blood digestion. However, in a paper by (Xavier et al. 2019) they found that this cathepsin L is the same protein as the previously known BmGTI (Boophilus microplus Gut Thrombin Inhibitor). As such, the role of transcript TC15655 may be in both digestion and in mediating anticlotting activity (Renard et al. 2000, 2002; Xavier et al. 2019), and remains to be tested. Lastly, a putative dipeptidyl peptidase 1-like-encoding transcript, also known as Cathepsin C, (Contig1379) was also upregulated in the vaccinated group. This transcript does contain a signal peptide, but it is most likely a lysosome targeting peptide. Targeting this enzyme via vaccination will be challenging due to its intracellular location and being part of a large protein family, which can compensate for loss of function. To date, one multicomponent vaccine containing recombinant tick Cathepsins B, C, D, L and legumain from I. ricinus has been evaluated in a rabbit model. Challenge of the vaccinated animals with I. ricinus ticks showed a slight decrease of engorged females, despite high antibody titres observed against all the antigens (Franta 2012). The presence of antibodies merely indicate that the proteins are antigenic when administered during vaccination, but as these proteins occur inside the digestive vesicles where they function at acidic pH, they are not accessible to the immune response elicited upon vaccination. The feasibility of vaccinating against intracellular proteins remains questionable and therefore not a priority for further evaluation. Targeting cathepsins using small drugs remains a viable route based on (a) the expansion of novel lysosomal-delivery mechanisms for targeted drug delivery, and (b) rational inhibitor design using protein crystal structures to target parasite specific protein features. This approach is currently exploited for a number of parasites, including Plasmodium spp. (Barber et al. 2021; Sojka et al. 2021), Trypanosome spp. (Alvarez et al. 2021) and Schistosome spp. (Mughal et al. 2022).
One cystatin-L2-like cysteine peptidase inhibitor (CV444905) was downregulated in ticks fed on vaccinated cattle. The role of this inhibitor in R. microplus midgut tissues remains unconfirmed. Two serine protease inhibitors (TC23621, TC20102) that belong to the Papilin protein family (and containing Kunitz domains) were downregulated. Their specific biological function(s) remain uncharacterized. All three of these protease inhibitors are predicted to have signal peptides and might be accessible to the immune system. However, all of them are also part of large protein families that may compensate for loss of function during vaccination and/or drug treatment.
In ticks, the process of vitellogenesis is induced by the blood meal and mediated by ecdysteroids (see next section). Vitellin (Vn) is the major yolk protein and serves as an important nutrient for embryonic development. It is processed from its precursor vitellogenin (Vg), a large phosphoglycoprotein produced in the fat body, midgut and ovaries of ticks (Donohue et al. 2008). From these tissues, Vg is released into the hemolymph from where it is taken up by the oocytes via receptor-mediated endocytosis. Once inside the oocytes Vg is converted to Vn (Xavier et al. 2018). Disruption of the uptake of Vg into oocytes via gene known-down studies resulted in reduced fecundity in R. microplus ticks, supporting the role of Vg in tick reproduction (Xavier et al. 2018). However, due to vitellogenin’s ability to sequester haem, it might also play a role in haem detoxification, preventing oxidative stress and subsequent tissue damage (Khalil et al. 2013). In ticks fed on vaccinated animals, two vitellogenin-2-like transcripts (TC23222 and TC21529) were significantly decreased in the midgut tissue. Furthermore, a vitellin-degrading cysteine endopeptidase (VTDCE; TC21162) was also significantly downregulated. VTDCE is a cathepsin L-like enzyme fist discovered in the eggs of R. microplus (Seixas et al. 2003), but is also localised in the midgut where it is synthesised and transported through the haemolymph to the developing oocytes (Seixas et al. 2010).
These observations are expected as it is known that Bm86 vaccination reduces the fecundity of ticks and disrupts bloodmeal uptake due to leakage into the haemolymph. The question as to whether vaccination against any of the latter transcripts are promising remains to be evaluated, but this approach will face some serious obstacles such as (a) the need for very high antibody titres within the host and the haemolymph of the tick to inhibit the very large number of Vg transcripts, (b) adequate transport of antibodies across the tick midgut barriers in the absence of a barrier disrupting mechanism(s) such as Bm86 vaccination, (c) stability of antibodies within the haemolymph, as IgG has been shown to be rapidly degraded (Benyakir 1989), (d) overcoming the tissue-mediated compensation mechanisms of expressing Vg-transcripts from an array of tissues, (e) antibody binding needs to block the function all VGs to affect tick fecundity, (f) lack of maintaining host immune memory to sustain a protective response, to name but a few. Targeting of the vitellogenin receptor to block receptor-mediated endocytosis of Vg into oocytes remains an option, but will require significant insight into the receptor structure to design tick-specific compounds (Mitchell et al. 2019).
A very interesting gate to parasite control via targeting vitellogenesis came to light in 2021 in a paper by Perdomo et al. (2021). In their study, human blood miRNAs were found to be transported into the fat body tissue of Aedes aegypti mosquitoes from where it regulated the expression of mosquito genes. Using artificial feeding with blood containing human miR-21-5p they showed that the miRNA positively regulates the expression of the vitellogenin gene. This study opens the door to targeting host miRNAs (or small regulatory molecules) to disrupt the bloodmeal-mediated activation of parasite gene expression (Perdomo et al. 2021). Also, when combining the expansion of RNA-based therapeutics such as miRNA agomirs (Zhang et al. 2021; Damase et al. 2021), and the increase in studies on the role of regulatory RNAs in parasite fecundity (Li et al. 2021; Zhang and Raikhel 2021) this field holds a lot of promise. It must be noted that insight into the mechanisms regulating gene expression in ticks are essential to fully exploit cutting-edge RNA and/or DNA therapeutics.
Ecdysteroid signalling pathway
Ecdysteroids and juvenile hormones (JHs) are the two major hormone families that drive development, moulting, and reproductive physiology in insects (Lenaerts et al. 2019). Both hormones mediate their functions via nuclear receptors that control the transcription of an array of genes. Ecdysteroid synthesis, including the active derivative 20-hydroxyecdysone (20E), starts with the uptake of cholesterol. This uptake can be mediated by cholesterol transporters such as Nieman Pick proteins (Xavier et al. 2021) or via lipoproteins that enter cells through receptor-mediated endocytosis (Miller and Bose 2011). Cholesterol is converted enzymatically to 7-dehydrocholesterol by the oxygenase enzyme neverland (nvd). The remainder of the ecdysteroidogenic pathway is mediated by a number of cytochrome P450 enzymes (a.k.a. the Halloween genes), which mediates a series of oxidation and hydroxylation steps to finally produce 20E. These enzymes include: Cyp307a1/spook (spo); Cyp307a2/spookier (spok); Cyp306a1/phantom (phm); Cyp302a1/disembodied (dib); Cyp315a1/shadow (sad); and Cyp314a1/shade (shd) (Niwa and Niwa 2016). Once 20E is produced, it acts via the heterodimeric ecdysone receptor complex, ecdysone receptor/ultraspiracle (EcR/USP), to affect gene expression. In 2012, co-localisation studies in Rhodnius prolixus cells furthermore indicated that the EcR co-localises with Hsp90, the immunophilin FKBP52, the light chain 1 of the motor protein dynein and microtubules (Vafopoulou and Steel 2012), all of which was deemed essential for the intact nucleocytoplasmic shuttling of EcR (Fig. 3). Under conditions that cause depolymerization of microtubules, there was a clear reduction in nuclear EcR and a concomitant increase in cytoplasmic EcR (Vafopoulou and Steel 2012). In 2013 Liu et al. showed that also in Helicoverpa armigera (Lepidoptera) the ultraspiracle protein interacts with Hsp90 and proposed the formation of the transcription complex containing 20E, EcR, USP1 (phosphorylated) and Hsp90 that recognise ecdysone response elements (Liu et al. 2013) (Fig. 3). In Ornithodoros moubata (Horigane et al. 2008, 2007; Connat et al. 1984; Ogihara et al. 2015), Amblyomma americanum (Palmer et al. 2002), and Haemaphysalis longicornis (Yang and Liu 2022) ticks, it has been shown that the ecdysteroid pathway is induced by blood feeding and that 20E is vital to tick fitness.
It must be noted that in insects, ecdysteroid synthesis occurs in a specialised endocrine organ called the prothoracic gland which ticks do not possess. Instead, in both argasid and ixodid ticks, ecdysone is released from epidermal cells from where they enter haemolymph and affect a number of other tissues/organs such as the ovaries (Reuben Kaufman 2007).
In this study, whole midgut tissue was dissected and used for RNA isolation, and as such the tissue would have contained epidermal cells along with other smaller structures associated with the midgut. From the differentially expressed gene information obtained, a putative Niemann Pick type C 1a (NPC1a) (TC20218) was found as upregulated. Niemann-Pick type C1 is a large membrane glycoprotein with mostly a late endosomal localization which functions in the processing and utilization of endocytosed cholesterol as well as intracellular regulation of cholesterol metabolism (Hu et al. 2020). Another transcript (T0009333) encoding a palmitoyltransferase ZDHHC17-like protein involved in lipoprotein transport and protein palmitoylation was also upregulated. As it is known that ticks require cholesterol and lipids from their bloodmeal, it is expected that proteins involved in cholesterol and lipid uptake is upregulated. With regards to the Halloween genes: Neverland, Phantom, Disembodied and Shadow was found as downregulated in ticks fed on vaccinated animals, but the statistical significance of the data is low. For Spook and Shade very low gene expression levels was found in ticks fed on vaccinated animals. As such, the expression of these genes need to be further analysed using qPCR and the levels of 20E determined in future. A transcript encoding a putative ecdysteroid receptor (EcR) (T0005046) and a putative chaperone Hsp90 (F1-2-A_Clone_20) was significantly upregulated in ticks that fed on vaccinated cattle. No data for USP were found. Based on (a) the downregulation of the Halloween Genes and associated 20E synthesis, and (b) severe disruption of the cytoskeleton as discussed below and (c) the downregulation of vitellogenin which is a 20E response element regulated gene (Elgendy et al. 2021; Zhu et al. 2007), we propose that the EcR remains cytoplasmic and does not translocate to the nucleus to activate transcription (Fig. 3). This model needs to be verified, as it may pave the way forward to understanding the cellular function of Bm86. Taking this work further, it is evident that targeting cholesterol metabolism, and specifically the Niemann Pick type protein(s) are promising drug targets (Fig. 3). This approach is gaining attention in parasite control, especially in Plasmodium (Istvan et al. 2019; Ressurreição and van Ooij 2021), ticks (Xavier et al. 2021; Cabezas-Cruz et al. 2019; Marchesini et al. 2021) and mites (Mani et al. 2022).
Tick energy metabolism
Ingestion of anti-Bm86 antibodies by feeding ticks, results in antibody binding to the midgut luminal surfaces, and in conjunction with complement, results in an apparent lysis of gut epithelial cells and leakage of the gut contents (Willadsen 1997). As the primary organ for blood meal digestion and nutrient acquisition, disruption of the integrity and normal functioning of the midgut tissues will have a ‘knock-on’ effect on normal metabolic processes, possibly leading to dysfunctional nutrient acquisition and depletion of energy stores (Fig. 4). In this study, the apparent effect of this disruption is at least partially supported by the decreased expression of transcripts encoding proteins involved in blood meal digestion (e.g., proteases and peptidases) and nutrient transport (e.g., ferritin) (Table 2). In energy metabolism, dietary glucose (stored as glycogen) is a primary source for immediate energy for both ixodid and argasid ticks, whereas lipids (stored as triglycerides) also have other roles including structural components (Alasmari and Wall 2021; Cabezas-Cruz et al. 2017; Oleaga et al. 2017).
As expected, several transcripts with putative functions in both carbohydrate and lipid transport and metabolism were differentially expressed in female ticks fed on vaccinated cattle (relative to controls) (Table 2). Two of these transcripts encoded proteins that were significantly upregulated in female ticks feeding on vaccinated cattle (logFC > 2), and included a putative glucose-6-phosphatase (Contig2278), as well as a putative glucose transporter of the solute carrier family 2 (T0000903). It is hypothesized that transcripts such as these may function as part of a compensatory or rescue mechanism, to obtain dietary glucose for down-stream metabolic processes. In regards to targeting such transcripts for tick vaccine development, there is unfortunately no data currently available. A peptide vaccine designed against a similar Anopheles stephensi glucose transporter (GLUT1), was able to reduce mosquito survival by 5% in vaccinated mice (Couto et al. 2017). To further illustrate the hurdle in vaccine development within this space, only a DNA vaccine directed against a Schistosoma japonicum triose-phosphate isomerase (an enzyme traditionally involved in gluconeogenesis), was tested in pigs that resulted in a significant ~ 48% reduction in adult worm burdens in vaccinated pigs (Zhu et al. 2006). Moreover, this mostly intracellular protein was shown to also be secreted to the surface of the fluke (Jimenez-Sandoval et al. 2020), therefore, enabling it to elicit an effective host antibody response. For R. microplus, in vitro tick triose-phosphate isomerase (rRmTIM) inhibition assays using murine-derived monoclonal antibodies, demonstrated a high degree of enzyme inhibition (> 48%) (Saramago et al. 2012). But no in vivo vaccine efficacy in animal infestation trials have been demonstrated to date to validate selection of such a target for vaccine development.
In contrast, current research appears to focus more on targeting transporters and other intracellular enzymes involved in energy metabolism for chemotherapeutic control, e.g., to block transmission of parasitic protists such as Plasmodium (Wang and Wang 2020), Trypanosoma spp. (Haanstra et al. 2017) and Leishmania spp. (Ortiz et al. 2017). Such enzymes are also being explored as targets for drug development against helminths (Jimenez-Sandoval et al. 2020), e.g., Schistosoma mansoni (Hulme et al. 2022). Therefore, transcripts identified in this study that are related to energy metabolism are considered poor targets for vaccine development, due to the intracellular origin of most of these targets and the concomitant poor extracellular exposure for host immune interactions to confer protection. However, results may still offer targets for small drug development as next generation acaricides.
Oxidative stress responses
Blood feeding and digestion is also a source of oxidative stress since free haem and iron, as well as other molecules, that are released following haemolysis can be toxic to the tick tissues (Sabadin et al. 2021; Citelli et al. 2007). Therefore, ticks should have a well-orchestrated oxidative stress response to protect themselves from the deleterious effects of these by-products (Paes et al. 2001; Graca-Souza et al. 2006; Citelli et al. 2007).
Free cytoplasmic ferric iron released during heme digestion is sequestered intracellularly by a class of storage protein, ferritin 1 (Fer1), whereas iron destined for peripheral tissues are transported from digestive cells throughout the hemolymph bound to a secreted ferritin 2 (Fer2) (Kopáček et al. 2018). A putative ferritin 1 (NP1774071) was downregulated in tick midguts that fed on the vaccinated test group (Table 2). Since no other similar proteins were up-regulated it could be hypothesized that the midgut cells were experiencing a buildup of cytoplasmic iron (i.e., iron overload), similar to findings from knock-down experiments in other tick species (e.g. Haemaphysalis longicornis) (Galay et al. 2014b). The ferritin 2 protein has been tested as a vaccine target, and homologous challenge trials in cattle showed a 64% and 72% vaccine efficacy against infestations of R. microplus and R. annulatus ticks, respectively (Hajdusek et al. 2010). The use of ferritin 1 as a vaccine antigen has only been demonstrated in a rabbit model against H. longicornis infestation with an efficacy of ~ 34% (Galay et al. 2014a). However, since both tick ferritin 1 and 2 share a high degree of identity, the efficacy observed may be due to antibody cross-reactivity and not specific reactivity towards the intracellular ferritin 1. Iron metabolism as a whole is yet to be explored for chemotherapeutics, as has been the case for various human disorders (Crielaard et al. 2017).
Several transcripts involved with the glutathione detoxification pathway (TC17253 and TC17897) were also significantly down-regulated (Table 2). Glutathione S-transferase is a known phase II detoxification enzyme involved in management of oxidative stress. These enzymes are highly expressed in tick tissues during tick feeding where they act as intracellular scavengers of free heme (Rodriguez-Valle et al. 2010; Kopáček et al. 2018), similarly to other blood-feeding parasites, including Haemonchus contortus (van Rossum et al. 2004) and Ancylostoma caninum (Zhan et al. 2005). Therefore, down-regulation of these proteins can support the notion that the midgut tissues of ticks feeding on the test group were experiencing increased oxidative stress. A compensation mechanism is, however, evident as a putative GST encoding transcript (TC17316) was significantly upregulated (Table 2). A study by Sabadin et al. (2017) showed > 80% sequence similarity of a GST sequence from H. longicornis ticks (GST-HI) to GST from R. appendiculatus and R. sanguineus, with multiple conserved antigenic sites. Vaccination with recombinant GST from H. longicornis showed 57% efficacy against R. microplus infestation, showing anti-GST cross-reactivity in both species (Parizi et al. 2011). Consequently, the product of this transcript could be considered for further investigation as an additional candidate for a multivalent vaccine.
Hypoxia-inducible factor (HIF)-1 is a key transcriptional regulator that promotes cellular adaptation to oxidative stress and hypoxia for key biological processes that include energy metabolism, apoptosis and cell survival (Movafagh et al. 2015). A putative Egl nine homolog 1 transcript (TC24883) encoding a putative hypoxia-inducible prolyl hydroxylase 2 (PHD-2) was significantly downregulated (logFC > 3) in the test group (Table 2). This enzyme hydroxylates a proline residue in the alpha subunit of the HIF-1 transcription factor to enable proteasomal destruction by the ubiquitin E3 ligase complex under normal oxygen tension (Jaakkola et al. 2001), whereas enzyme function is inhibited by low oxygen concentrations or hypoxia (Stowe and Camara 2009). As a result of an increase in pro-oxidants (e.g., Fe(II) and heme), a hypoxic environment could be created within the tick midgut tissues that inhibits the function of PHD-2. Consequently, HIF-1 alpha is not degraded by the proteasome following ubiquitination and therefore proceeds to form a transcription complex with the HIF-1 beta subunit which then acts as a master regulator of numerous hypoxia-inducible genes (Greer et al. 2012). This observation is further substantiated by the concomitant decreased abundance of transcripts (i.e., CK184995) that have putative functions in ubiquitination and perhaps degradation of HIF-1 alpha (Kamura et al. 2000; Tandle et al. 2009). For additional support, a putative selenium-binding protein (TC16153) was also down-regulated that has been implicated to be a negative regulator of HIF-1 alpha during recovery from injury and cancer progression in humans (Jeong et al. 2014; Seelig et al. 2021). However, this function has not been described in arthropods to date and can be tested further in future studies.
Transcripts encoding the HIF-1 regulator molecule were identified in this study to be non-differentially expressed (data not shown), this is expected as it has been shown that HIF-1 in humans is constitutively expressed (Ke and Costa, 2006). Increased levels of Hsp90 and Hsp70 (Genin et al. 2008), stabilises HIF-1 alpha and protects it from degradation by the proteasome (Zhou et al. 2004). Putative Hsp70 (T0000364) and Hsp90 (F1-2-A_Clone_20) encoding transcripts were upregulated in ticks feeding on vaccinated hosts. The inhibition of a putative PHD-2 under hypoxic conditions has also been associated with cytoskeletal remodelling assisting in translocation of HIF-1 alpha to the nucleus, where it can associate with HIF-1 beta to form the functional transcription factor (Guo et al. 2017; Weidemann et al. 2013). In this regard, two putative alpha-tubulin encoding transcripts (T0004546 and AA257910) were significantly upregulated (logFC > 2) (Table 2). In response to oxygen-depletion, HIF-1 signalling can regulate glucose uptake and anaerobic responses by activating the transcription of glucose transporter 1 (GLUT1) (Chen et al. 2001), as shown by the upregulation of a GLUT1 encoding transcript (T0000903) (Table 2).
Recent evidence also suggests that hypoxic stress can suppress Notch activity, involved in cell-to-cell communication during tissue development, and expression of its downstream signalling molecules (Itoh et al. 2019). In this regard, two transcripts encoding putative proteins involved in the Notch signalling pathway were downregulated in this study (i.e., TC23300 and TC18726) (Table 2). Consequently, an alternative pathway to promote cell growth and differentiation may be up-regulated. This hypothesis is based on the up-regulation of a putative epithelial growth factor, granulin (TC16526), which has been shown in mice to induce DNA synthesis and stimulate cell growth by activating mitogen-activating protein kinase (MAPK) and the phosphatidylinositol 3-kinase (PI-3 K) pathways (Zanocco-Marani et al. 1999). Activation of these pathways lead to cell growth and activation of crucial metabolic functions such as synthesis of lipids, proteins and glycogen (Lodish et al. 2008).
Taken together, the data identifies possible metabolic pathways that serve as a rescue mechanism explaining why ticks can survive even after vaccination, which disrupts feeding and growth. Only a granulin-like growth factor (Ov-GRN-1) from the fluke Opisthorchis viverrini has been considered for further development as a potential vaccine candidate (McManus 2020). However, the utility of this target as a tick vaccine antigen remains to be demonstrated. Intracelluar targets such as ubiquitin (UBQ) and elongation factor 1 alpha (EF1a), have been tested in cattle vaccination and R. microplus infestation trials, with 0% and 38% vaccine efficacies reported, respectively (Almazán et al. 2012). No other targets of the HIF-1 alpha pathway have been successfully exploited for antiparasitic vaccine development and since the majority of these targets are intracellular, these may rather be pursued as targets for future drug development.
Innate immune system
During parasite feeding, ticks can be subjected to pathogens in the host’s blood and/or which reside in the tick gut (van Oosterwijk and Wikel 2021). Several transcripts that have a putative role in the tick immune system were significantly downregulated (logFC > 2) in the midgut tissue of ticks that fed on vaccinated cattle. These include putative ixoderins (CK189663, TC22409 and TC19369) and the antimicrobial peptide microplusin (TC23502) (Table 2). The continuing co-evolution of ticks with their associated pathogens promotes favourable characteristics of tick physiology to successfully maintain and transmit pathogens without compromising the fitness of the tick vector (Mans 2011). Ticks feeding on vaccinated cattle seem to also have a compromised innate immunity, and it is hypothesised that the Bm86 and RmAg3 proteins may function synergistically in vivo to protect the tick from the damaging effects of its ingested microbes. Disruption of the innate immune system of ticks could potentially prevent the successful colonisation and eventual transmission of microbes to the host and also represents a target for transmission blocking vaccines (van Oosterwijk and Wikel 2021). This has in principle been proven for cattle vaccinated with Bm86, which reduced the transmission of Anaplasma marginale by reducing the number of infected ticks (de la Fuente et al. 1998). However, targeting tick innate immunity per se for tick control, remains to be validated as no vaccines successfully targeting this aspect of tick biology exist. Similarly, targeting innate immunity for antiparasitic drugs has also not been demonstrated to date. However, tick-derived antimicrobial peptides have been explored as treatments for human microbial infections, e.g., microplusin against Cryptococcus neoformans (Silva et al. 2011).
Conclusions
Multicomponent vaccines have enjoyed a lot of attention over the past two decades in the development of vaccines against complex parasites. Examples include, but are not limited to vaccines against parasitic worms such as the nematode Teladorsagia circumcincta (Nisbet et al. 2013), tapeworms (Taenia saginata) (Lightowlers et al. 1996), mites (Dermanyssus gallinae) (Bartley et al. 2012) and ticks (Ndawula and Tabor 2020). Multicomponent vaccines that target the various life stages of Plasmodium falciparum is also well documented such as vaccines that target multiple steps in the erythrocyte invasion pathway (Bustamante et al. 2017), multiple developmental stages (Boes et al. 2015), and protein–protein interactions (PPIs) within P. yoelli (Spring et al. 2009; Ouattara et al. 2010; Thera et al. 2011). The development of multicomponent vaccines for ticks will require an in-depth understanding of tick biology and the complex interaction of responses that occur during the whole life cycle, and in response to current vaccines on the market. Criteria for the selection of multicomponent vaccines following expression profiling remains challenging, as insight into the effects of concentration of antigen expression; timing and length of expression in the parasite and subcellular location, remains unknown. Clearly there are many questions to deal with in this area.
To date, vaccines against complex parasites fail to offer complete protection, and as such the combination of vaccines with small drugs (anti-parasitic drugs and acaricides) remains at the forefront. With the increase in acaricide resistance, new targets for chemical control is in dire need. This include the development of new acaricides and/or small drugs. Development of new therapeutics is also attractive as small drugs can potentially target numerous tick species, and have potentially lower production costs. High-throughput in silico drug discovery can be attempted, even in the absence of crystal structure data. Tools such as AlphaFold (Jumper et al. 2021) will enable ab initio prediction of high quality target protein structure models that can be used in drug docking studies. In summary, this paper provides a number of targets for the future development of vaccines and small molecular therapeutics, as well as novel pathways that can be targeted towards improving the outcome on tick control.
Data availability
Normalized microarray data was submitted to the GEO repository of the National Centre for Biotechnology Information on accession no GSE221646.
References
Agbede R, Kemp D (1986) Immunization of cattle against Boophilus microplus using extracts derived from adult female ticks: histopathology of ticks feeding on vaccinated cattle. Int J Parasitol 16:35–41
Alasmari S, Wall R (2021) Metabolic rate and resource depletion in the tick Ixodes ricinus in response to temperature. Exp Appl Acarol 83:81–93
Almazán C, Kocan KM, Bergman DK, Garcia-Garcia JC, Blouin EF, De La Fuente J (2003) Identification of protective antigens for the control of Ixodes scapularis infestations using cDNA expression library immunization. Vaccine 21:1492–1501
Almazán C, Moreno-Cantú O, Moreno-Cid JA, Galindo RC, Canales M, Villar M, De La Fuente J (2012) Control of tick infestations in cattle vaccinated with bacterial membranes containing surface-exposed tick protective antigens. Vaccine 30:265–272
Alvarez VE, Iribarren PA, Niemirowicz GT, Cazzulo JJ (2021) Update on relevant trypanosome peptidases: validated targets and future challenges. Biochim Biophys Acta 1869:140577
Atchou K, Ongus J, Machuka E, Juma J, Tiambo C, Djikeng A, Silva JC, Pelle R (2020) Comparative transcriptomics of the bovine apicomplexan parasite Theileria parva developmental stages reveals massive gene expression variation and potential vaccine antigens. Front Vet Sci 7:287
Barber J, Sikakana P, Sadler C, Baud D, Valentin JP, Roberts R (2021) A target safety assessment of the potential toxicological risks of targeting plasmepsin IX/X for the treatment of malaria. Toxicol Res 10:203–213
Barrero RA, Guerrero FD, Black M, Mccooke J, Chapman B, Schilkey F, de León AAP, Miller RJ, Bruns S, Dobry J (2017) Gene-enriched draft genome of the cattle tick Rhipicephalus microplus: assembly by the hybrid Pacific Biosciences/Illumina approach enabled analysis of the highly repetitive genome. Int J Parasitol 47:569–583
Bartley K, Huntley JF, Wright HW, Nath M, Nisbet AJ (2012) Assessment of cathepsin D and L-like proteinases of poultry red mite, Dermanyssus gallinae (De Geer), as potential vaccine antigens. Parasitology 139:755–765
Benyakir D (1989) Quantitative studies of host immunoglobulin-g in the hemolymph of ticks (ACARI). J Med Entomol 26:243–246
Blecha IMZ, Csordas BG, Aguirre AAR, Cunha RC, Garcia MV, Andreotti R (2018) Analysis of Bm86 conserved epitopes: is a global vaccine against Cattle Tick Rhipicephalus microplus possible? Rev Bras Parasitol Vet 27:267–279
Boes A, Spiegel H, Voepel N, Edgue G, Beiss V, Kapelski S, Fendel R, Scheuermayer M, Pradel G, Bolscher JM, Behet MC, Dechering KJ, Hermsen CC, Sauerwein RW, Schillberg S, Reimann A, Fischer R (2015) Analysis of a multi-component multi-stage malaria vaccine candidate-tackling the cocktail challenge. PLoS ONE 10:e0131456
Bustamante LY, Powell GT, Lin Y-C, Macklin MD, Cross N, Kemp A, Cawkill P, Sanderson T, Crosnier C, Muller-Sienerth N, Doumbo OK, Traore B, Crompton PD, Cicuta P, Tran TM, Wright GJ, Rayner JC (2017) Synergistic malaria vaccine combinations identified by systematic antigen screening. Proc Natl Acad Sci 114:12045–12050
Cabezas-Cruz A, Alberdi P, Valdés JJ, Villar M, de la Fuente J (2017) Anaplasma phagocytophilum infection subverts carbohydrate metabolic pathways in the tick vector, Ixodes scapularis. Front Cell Infect Microbiol 7:23
Cabezas-Cruz A, Espinosa P, Alberdi P, de la Fuente J (2019) Tick-pathogen interactions: the metabolic perspective. Trends Parasitol 35:316–328
Cerqueira APM, Santana IB, Araújo JSC, Lima HG, Batatinha MJM, Branco A, Santos Junior MCD, Botura MB (2022) Homology modeling, docking, molecular dynamics and in vitro studies to identify Rhipicephalus microplus acetylcholinesterase inhibitors. J Biomol Struct Dyn 40:6787–6797
Chen C, Pore N, Behrooz A, Ismail-Beigi F, Maity A (2001) Regulation of glut1 mRNA by hypoxia-inducible factor-1. Interaction between H-ras and hypoxia. J Biol Chem 276:9519–9525
Cimermancic P, Weinkam P, Rettenmaier TJ, Bichmann L, Keedy DA, Woldeyes RA, Schneidman-Duhovny D, Demerdash ON, Mitchell JC, Wells JA, Fraser JS, Sali A (2016) CryptoSite: expanding the druggable proteome by characterization and prediction of cryptic binding sites. J Mol Biol 428:709–719
Citelli M, Lara FA, da Silva Vaz I, Oliveira PL (2007) Oxidative stress impairs heme detoxification in the midgut of the cattle tick, Rhipicephalus (Boophilus) microplus. Mol Biochem Parasitol 151:81–88
Coetzer J, Tustin R (2004) Infectious diseases of livestock, vol 2. Oxford University Press, Cape Town
Conesa A, Götz S, García-Gómez JM, Terol J, Talón M, Robles M (2005) Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21:3674–3676
Connat JL, Diehl PA, Morici M (1984) Metabolism of ecdysteroids during the vitellogenesis of the tick Ornithodoros moubata (Ixodoidea, Argasidae): accumulation of apolar metabolites in the eggs. Gen Comp Endocrinol 56:100–110
Costa GCA, Ribeiro ICT, Melo O, Gontijo NF, Sant’anna MRV, Pereira MH, Pessoa GCD, Koerich LB, Oliveira F, Valenzuela JG, Giunchetti RC, Fujiwara RT, Bartholomeu DC, Araujo RN (2021) Amblyomma sculptum salivary protease inhibitors as potential anti-tick vaccines. Front Immunol 11:611104
Couto J, Antunes S, Ferrolho J, de la Fuente J, Domingos A (2017) Reduction of mosquito survival in mice vaccinated with Anopheles stephensi glucose transporter. Biomed Res Int 2017:3428186
Crielaard BJ, Lammers T, Rivella S (2017) Targeting iron metabolism in drug discovery and delivery. Nat Rev Drug Discov 16:400–423
Cruz CE, Fogaça AC, Nakayasu ES, Angeli CB, Belmonte R, Almeida IC, Miranda A, Miranda MTM, Tanaka AS, Braz GR (2010) Characterization of proteinases from the midgut of Rhipicephalus (Boophilus) microplus involved in the generation of antimicrobial peptides. Parasit Vectors 3:63
Cunha RC, Andreotti R, Garcia MV, Aguirre ADR, Leitao A (2013) Calculation of the efficacy of vaccines against tick infestations on cattle. Revista Brasileira De Parasitologia Veterinaria 22:571–578
Damase TR, Sukhovershin R, Boada C, Taraballi F, Pettigrew RI, Cooke JP (2021) The limitless future of RNA therapeutics. Front Bioeng Biotechnol 9:161
de la Fuente J, Rodríguez M, Redondo M, Montero C, García-García JC, Méndez L, Serrano E, Valdés M, Enriquez A, Canales M, Ramos E, Boué O, Machado H, Lleonart R, de Armas CA, Rey S, Rodríguez JL, Artiles M, García L (1998) Field studies and cost-effectiveness analysis of vaccination with Gavac against the cattle tick Boophilus microplus. Vaccine 16:366–373
de la Fuente J, Almazán C, Canales M, Pérez de la Lastra JM, Kocan KM, Willadsen P (2007) A ten-year review of commercial vaccine performance for control of tick infestations on cattle. Anim Health Res Rev 8(1):23–28. https://doi.org/10.1017/S1466252307001193
Donohue K, Khalil S, Mitchell R, Sonenshine D, Michael Roe R (2008) Molecular characterization of the major hemelipoglycoprotein in ixodid ticks. Insect Mol Biol 17:197–208
Dzemo WD, Thekisoe O, Vudriko P (2022) Development of acaricide resistance in tick populations of cattle: a systematic review and meta-analysis. Heliyon 8:e08718
Elgendy AM, Mohamed AA, Duvic B, Tufail M, Takeda M (2021) Involvement of cis-acting elements in molecular regulation of JH-mediated vitellogenin gene 2 of female Periplaneta americana. Front Physiol 12:723072
Fauman EB, Rai BK, Huang ES (2011) Structure-based druggability assessment–identifying suitable targets for small molecule therapeutics. Curr Opin Chem Biol 15:463–468
Fogaça AC, da Silva PI, Miranda MTM, Bianchi AG, Miranda A, Ribolla PE, Daffre S (1999) Antimicrobial activity of a bovine hemoglobin fragment in the tick Boophilus microplus. J Biol Chem 274:25330–25334
Franta Z (2012) Experimental vaccinations of rabbits with recombinant digestive peptidases of the tick Ixodes ricinus. Ph.D. Masters, University of South Bohemia
Galay RL, Miyata T, Umemiya-Shirafuji R, Maeda H, Kusakisako K, Tsuji N, Mochizuki M, Fujisaki K, Tanaka T (2014a) Evaluation and comparison of the potential of two ferritins as anti-tick vaccines against Haemaphysalis longicornis. Parasit Vectors 7:482
Galay RL, Umemiya-Shirafuji R, Bacolod ET, Maeda H, Kusakisako K, Koyama J, Tsuji N, Mochizuki M, Fujisaki K, Tanaka T (2014b) Two kinds of ferritin protect ixodid ticks from iron overload and consequent oxidative stress. PLoS ONE 9:e90661
Garcia GR, Chaves Ribeiro JM, Maruyama SR, Gardinassi LG, Nelson K, Ferreira BR, Andrade TG, de Miranda Santos IKF (2020) A transcriptome and proteome of the tick Rhipicephalus microplus shaped by the genetic composition of its hosts and developmental stage. Sci Rep 10:12857
Genin O, Hasdai A, Shinder D, Pines M (2008) Hypoxia, Hypoxia-Inducible Factor-1α (HIF-1α), and heat-shock proteins in tibial dyschondroplasia. Poult Sci 87:1556–1564
Graca-Souza AV, Maya-Monteiro C, Paiva-Silva GO, Braz GR, Paes MC, Sorgine MH, Oliveira MF, Oliveira PL (2006) Adaptations against heme toxicity in blood-feeding arthropods. Insect Biochem Mol Biol 36:322–335
Greer SN, Metcalf JL, Wang Y, Ohh M (2012) The updated biology of hypoxia-inducible factor. EMBO J 31:2448–2460
Guo H, Zheng H, Wu J, Ma H-P, Yu J, Yiliyaer M (2017) The key role of microtubules in hypoxia preconditioning-induced nuclear translocation of HIF-1α in rat cardiomyocytes. PeerJ 5:e3662–e3662
Haanstra JR, Gerding A, Dolga AM, Sorgdrager FJH, Buist-Homan M, du Toit F, Faber KN, Holzhütter HG, Szöör B, Matthews KR, Snoep JL, Westerhoff HV, Bakker BM (2017) Targeting pathogen metabolism without collateral damage to the host. Sci Rep 7:40406
Hajdusek O, Almazán C, Loosova G, Villar M, Canales M, Grubhoffer L, Kopacek P, de la Fuente J (2010) Characterization of ferritin 2 for the control of tick infestations. Vaccine 28:2993–2998
Hellemans J, Mortier G, de Paepe A, Speleman F, Vandesompele J (2007) qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol 8:1–14
Horigane M, Ogihara K, Nakajima Y, Shinoda T, Taylor D (2007) Cloning and expression of the ecdysteroid receptor during ecdysis and reproduction in females of the soft tick, Ornithodoros moubata (Acari: Argasidae). Insect Mol Biol 16:601–612
Horigane M, Ogihara K, Nakajima Y, Taylor D (2008) Isolation and expression of the retinoid X receptor from last instar nymphs and adult females of the soft tick Ornithodoros moubata (Acari: Argasidae). Gen Comp Endocrinol 156:298–311
Hu E, Meng Y, Ma Y, Song R, Hu Z, Li M, Hao Y, Fan X, Wei L, Fan S, Chen S, Zhai X, Li Y, Zhang W, Zhang Y, Guo Q, Bayin C (2020) De novo assembly and analysis of the transcriptome of the Dermacentor marginatus genes differentially expressed after blood-feeding and long-term starvation. Parasit Vectors 13:563
Hulme BJ, Geyer KK, Forde-Thomas JE, Padalino G, Phillips DW, Ittiprasert W, Karinshak SE, Mann VH, Chalmers IW, Brindley PJ, Hokke CH, Hoffmann KF (2022) Schistosoma mansoni α-N-acetylgalactosaminidase (SmNAGAL) regulates coordinated parasite movement and egg production. PLoS Pathog 18:e1009828
Istvan ES, Das S, Bhatnagar S, Beck JR, Owen E, Llinas M, Ganesan SM, Niles JC, Winzeler E, Vaidya AB, Goldberg DE (2019) Plasmodium Niemann-Pick type C1-related protein is a druggable target required for parasite membrane homeostasis. Elife 8:e40529
Itoh M, Okuhashi Y, Takahashi Y, Sonoda Y, Mohammad S, Saito T, Shiratori E, Tohda S (2019) Hypoxia up-regulates HIF expression while suppressing cell growth and Notch activity in leukaemia cells. Anticancer Res 39:4165–4170
Jaakkola P, Mole DR, Tian Y-M, Wilson MI, Gielbert J, Gaskell SJ, von Kriegsheim A, Hebestreit HF, Mukherji M, Schofield CJ (2001) Targeting of HIF-α to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science 292:468–472
Jalal K, Abu-Izneid T, Khan K, Abbas M, Hayat A, Bawazeer S, Uddin R (2022) Identification of vaccine and drug targets in Shigella dysenteriae sd197 using reverse vaccinology approach. Sci Rep 12:251
Jeong JY, Zhou JR, Gao C, Feldman L, Sytkowski AJ (2014) Human selenium binding protein-1 (hSP56) is a negative regulator of HIF-1α and suppresses the malignant characteristics of prostate cancer cells. BMB Rep 47:411–416
Jia N, Wang J, Shi W, Du L, Sun Y, Zhan W, Jiang J-F, Wang Q, Zhang B, Ji P (2020) Large-scale comparative analyses of tick genomes elucidate their genetic diversity and vector capacities. Cell 182:1328–1340
Jimenez-Sandoval P, Castro-Torres E, González-González R, Díaz-Quezada C, Gurrola M, Camacho-Manriquez LD, Leyva-Navarro L, Brieba LG (2020) Crystal structures of Triosephosphate isomerases from Taenia solium and Schistosoma mansoni provide insights for vaccine rationale and drug design against helminth parasites. PLoS Negl Trop Dis 14:e0007815
Jumper J, Evans R, Pritzel A, Green T, Figurnov M, Ronneberger O, Tunyasuvunakool K, Bates R, Žídek A, Potapenko A, Bridgland A, Meyer C, Kohl SAA, Ballard AJ, Cowie A, Romera-Paredes B, Nikolov S, Jain R, Adler J, Back T, Petersen S, Reiman D, Clancy E, Zielinski M, Steinegger M, Pacholska M, Berghammer T, Bodenstein S, Silver D, Vinyals O, Senior AW, Kavukcuoglu K, Kohli P, Hassabis D (2021) Highly accurate protein structure prediction with AlphaFold. Nature 596:583–589
Kamura T, Sato S, Iwai K, Czyzyk-Krzeska M, Conaway RC, Conaway JW (2000) Activation of HIF1α ubiquitination by a reconstituted von Hippel-Lindau (VHL) tumor suppressor complex. Proc Natl Acad Sci 97:10430–10435
Khalil SM, Donohue KV, Roe RM, Sonenshine DE (2013) Heme-binding lipoglyco storage proteins. Biol Ticks 1:398–415
Kopáček P, Perner J, Sojka D, Šíma R, Hajdušek O (2018) Molecular targets to impair blood meal processing in ticks. Ectoparasites
Kozakov D, Hall DR, Napoleon RL, Yueh C, Whitty A, Vajda S (2015) New frontiers in druggability. J Med Chem 58:9063–9088
Lenaerts C, Marchal E, Peeters P, Vanden Broeck J (2019) The ecdysone receptor complex is essential for the reproductive success in the female desert locust, Schistocerca gregaria. Sci Rep 9:15
Li LY, Wang S, Huang KY, Zhang YT, Li YL, Zhang M, Huang JY, Deng ZY, Ni XZ, Li XC (2021) Identification and characterization of MicroRNAs in Gonads of Helicoverpa armigera (Lepidoptera: Noctuidae). InSects 12:749
Lightowlers MW, Rolfe R, Gauci CG (1996) Taenia saginata: vaccination against cysticercosis in cattle with recombinant oncosphere antigens. Exp Parasitol 84:330–338
Lipinski CA, Lombardo F, Dominy BW, Feeney PJ (1997) Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv Drug Deliv Rev 23:3–25
Liu W, Zhang F-X, Cai M-J, Zhao W-L, Li X-R, Wang J-X, Zhao X-F (2013) The hormone-dependent function of Hsp90 in the crosstalk between 20-hydroxyecdysone and juvenile hormone signaling pathways in insects is determined by differential phosphorylation and protein interactions. Biochem Biophys Acta 1830:5184–5192
Lodish HF, Berk A, Kaiser CA, Krieger M, Cscott MP, Bretscher A, Ploegh H (2008) Molecular cell biology, 6th edn. WH Freeman, New York
Lovis L, Reggi J, Berggoetz M, Betschart B, Sager H (2013) Determination of acaricide resistance in Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) field populations of Argentina, South Africa, and Australia with the larval tarsal test. J Med Entomol 50:326–335
Maizels RM (2021) Identifying novel candidates and configurations for human helminth vaccines. Expert Rev Vaccines 20:1389–1393
Mani K, Nganso BT, Rodin P, Otmy A, Rafaeli A, Soroker V (2022) Effects of Niemann-Pick type C2 (NPC2) gene transcripts silencing on behavior of Varroa destructor and molecular changes in the putative olfactory gene networks. Insect Biochem Mol Biol 148:103817
Mans BJ (2011) Evolution of vertebrate hemostatic and inflammatory control mechanisms in blood-feeding arthropods. J Innate Immun 3:41–51
Marchesini P, Lemos ASO, Bitencourt ROB, Fiorotti J, Angelo IDC, Fabri RL, Costa-Júnior LM, Lopes WDZ, Bittencourt V, Monteiro C (2021) Assessment of lipid profile in fat body and eggs of Rhipicephalus microplus engorged females exposed to (E)-cinnamaldehyde and α-bisabolol, potential acaricide compounds. Vet Parasitol 300:109596
Maritz-Olivier C, van Zyl W, Stutzer C (2012) A systematic, functional genomics, and reverse vaccinology approach to the identification of vaccine candidates in the cattle tick, Rhipicephalus microplus. Ticks Tick Borne Dis 3:179–187
Mcmanus DP (2020) Recent progress in the development of liver fluke and blood fluke vaccines. Vaccines 8:553
Miller WL, Bose HS (2011) Early steps in steroidogenesis: intracellular cholesterol trafficking. J Lipid Res 52:2111–2135
Mistry J, Chuguransky S, Williams L, Qureshi M, Salazar GA, Sonnhammer ELL, Tosatto SCE, Paladin L, Raj S, Richardson LJ, Finn RD, Bateman A (2020) Pfam: the protein families database in 2021. Nucleic Acids Res 49:D412–D419
Mitchell RD, Sonenshine DE, de Leon AAP (2019) Vitellogenin receptor as a target for tick control: a mini-review. Front Physiol 10:618
Movafagh S, Crook S, Vo K (2015) Regulation of hypoxia-inducible factor-1a by reactive oxygen species: new developments in an old debate. J Cell Biochem 116:696–703
Moxon R, Reche PA, Rappuoli R (2019) Editorial: reverse vaccinology. Front Immunol 10:618
Mughal MNN, Grevelding CGG, Haeberlein S (2022) The anticancer drug imatinib induces autophagy in Schistosoma mansoni. Int J Parasitol 52:211–215
Muhanguzi D, Byaruhanga J, Amanyire W, Ndekezi C, Ochwo S, Nkamwesiga J, Mwiine FN, Tweyongyere R, Fourie J, Madder M (2020) Invasive cattle ticks in East Africa: morphological and molecular confirmation of the presence of Rhipicephalus microplus in south-eastern Uganda. Parasit Vectors 13:1–9
Ndawula C, Tabor AE (2020) Cocktail anti-tick vaccines: the unforeseen constraints and approaches toward enhanced efficacies. Vaccines 8:457
Nijhof AM, Balk JA, Postigo M, Jongejan F (2009) Selection of reference genes for quantitative RT-PCR studies in Rhipicephalus (Boophilus) microplus and Rhipicephalus appendiculatus ticks and determination of the expression profile of Bm86. Bmc Mol Biol 10:1–14
Nisbet AJ, Mcneilly TN, Wildblood LA, Morrison AA, Bartley DJ, Bartley Y, Longhi C, Mckendrick IJ, Palarea-Albaladejo J, Matthews JB (2013) Successful immunization against a parasitic nematode by vaccination with recombinant proteins. Vaccine 31:4017–4023
Niwa YS, Niwa R (2016) Transcriptional regulation of insect steroid hormone biosynthesis and its role in controlling timing of molting and metamorphosis. Dev Growth Differ 58:94–105
Nyangiwe N, Harrison A, Horak IG (2013) Displacement of Rhipicephalus decoloratus by Rhipicephalus microplus (Acari: Ixodidae) in the Eastern Cape Province, South Africa. Exp Appl Acarol 61:371–382
Nyangiwe N, Horak IG, van der Mescht L, Matthee S (2017) Range expansion of the economically important Asiatic blue tick, Rhipicephalus microplus, in South Africa. J S Afr Vet Assoc 88:7
Ogihara MH, Hikiba J, Suzuki Y, Taylor D, Kataoka H (2015) Ovarian ecdysteroidogenesis in both immature and mature stages of an Acari, Ornithodoros moubata. PLoS ONE 10:e0124953
Oleaga A, Obolo-Mvoulouga P, Manzano-Román R, Pérez-Sánchez R (2017) A proteomic insight into the midgut proteome of Ornithodoros moubata females reveals novel information on blood digestion in argasid ticks. Parasit Vectors 10:366
Ortiz D, Guiguemde WA, Hammill JT, Carrillo AK, Chen Y, Connelly M, Stalheim K, Elya C, Johnson A, Min J, Shelat A, Smithson DC, Yang L, Zhu F, Guy RK, Landfear SM (2017) Discovery of novel, orally bioavailable, antileishmanial compounds using phenotypic screening. PLoS Negl Trop Dis 11:e0006157
Osterloh A (2022) Vaccination against bacterial infections: challenges, progress, and new approaches with a focus on intracellular bacteria. Vaccines 10:751
Ouattara A, Mu J, Takala-Harrison S, Saye R, Sagara I, Dicko A, Niangaly A, Duan J, Ellis RD, Miller LH (2010) Lack of allele-specific efficacy of a bivalent AMA1 malaria vaccine. Malar J 9:175
Paes MC, Oliveira MB, Oliveira PL (2001) Hydrogen peroxide detoxification in the midgut of the blood-sucking insect, Rhodnius prolixus. Arch Insect Biochem Physiol 48:63–71
Palmer MJ, Warren JT, Jin X, Guo X, Gilbert LI (2002) Developmental profiles of ecdysteroids, ecdysteroid receptor mRNAs and DNA binding properties of ecdysteroid receptors in the Ixodid tick Amblyomma americanum (L.). Insect Biochem Mol Biol 32(4):465–476. https://doi.org/10.1016/s0965-1748(01)00124-2
Parizi LF, Utiumi KU, Imamura S, Onuma M, Ohashi K, Masuda A, da Silva Vaz I Jr (2011) Cross immunity with Haemaphysalis longicornis glutathione S-transferase reduces an experimental Rhipicephalus (Boophilus) microplus infestation. Exp Parasitol 127:113–118
Perdomo HD, Hussain M, Parry R, Etebari K, Hedges LM, Zhang GM, Schulz BL, Asgari S (2021) Human blood microRNA hsa-miR-21–5p induces vitellogenin in the mosquito Aedes aegypti. Commun Biol 4:856
Pereira DFS, Ribeiro HS, Gonçalves AAM, da Silva AV, Lair DF, de Oliveira DS, Boas DFV, Conrado IDSS, Leite JC, Barata LM, Reis PCC, Mariano RMDS, Santos TAP, Coutinho DCO, Gontijo NDF, Araujo RN, Galdino AS, Paes PRDO, Melo MM, Nagem RAP, Dutra WO, Silveira-Lemos DD, Rodrigues DS, Giunchetti RC (2022) Rhipicephalus microplus: an overview of vaccine antigens against the cattle tick. Ticks Tick-Borne Dis 13:101828
Pirahmadi S, Afzali S, Zargar M, Zakeri S, Mehrizi AA (2021) How can we develop an effective subunit vaccine to achieve successful malaria eradication? Microb Pathog 160:105203
Rathinasamy V, Poole WA, Bastos RG, Suarez CE, Cooke BM (2019) Babesiosis vaccines: lessons learned, challenges ahead, and future glimpses. Trends Parasitol 35:622–635
Renard G, Garcia JF, Cardoso FC, Richter MF, Sakanari JA, Ozaki LS, Termignoni C, Masuda A (2000) Cloning and functional expression of a Boophilus microplus cathepsin L-like enzyme. Insect Biochem Mol Biol 30:1017–1026
Renard G, Lara FA, de Cardoso FC, Miguens FC, Dansa-Petretski M, Termignoni C, Masuda A (2002) Expression and immunolocalization of a Boophilus microplus cathepsin L-like enzyme. Insect Mol Biol 11:325–328
Ressurreição M, van Ooij C (2021) Lipid transport proteins in malaria, from Plasmodium parasites to their hosts. Biochim Biophys Acta Mol Cell Biol Lipids 1866:159047
Reuben Kaufman W (2007) Gluttony and sex in female ixodid ticks: how do they compare to other blood-sucking arthropods? J Insect Physiol 53:264–273
Rodriguez-Valle M, Lew-Tabor A, Gondro C, Moolhuijzen P, Vance M, Guerrero FD, Bellgard M, Jorgensen W (2010) Comparative microarray analysis of Rhipicephalus (Boophilus) microplus expression profiles of larvae pre-attachment and feeding adult female stages on Bos indicus and Bos taurus cattle. BMC Genomics 11:437
Rodriguez-Vivas RI, Jonsson NN, Bhushan C (2018) Strategies for the control of Rhipicephalus microplus ticks in a world of conventional acaricide and macrocyclic lactone resistance. Parasitol Res 117:3–29
Saaid AA, Salih DA, Elhaj LM, Abdalla MA, Baumann M, Obara I, Ahmed JS, Clausen PH, El Hussein ARM (2020) The protection afforded to cattle immunized with Theileria annulata infected cell line is enhanced by subunit vaccine candidate TaSP. Transbound Emerg Dis 67:26–34
Sabadin GA, Parizi LF, Kiio I, Xavier MA, da Silva Matos R, Camargo-Mathias MI, Githaka NWO, Nene V, da Silva Vaz I (2017) Effect of recombinant glutathione S-transferase as vaccine antigen against Rhipicephalus appendiculatus and Rhipicephalus sanguineus infestation. Vaccine 35:6649–6656
Sabadin GA, Salomon TB, Leite MS, Benfato MS, Oliveira PL, da Silva Vaz I Jr (2021) An insight into the functional role of antioxidant and detoxification enzymes in adult Rhipicephalus microplus female ticks. Parasitol Int 81:102274
Saramago L, Franceschi M, Logullo C, Masuda A, Vaz Ida S, Farias SE, Moraes J (2012) Inhibition of enzyme activity of Rhipicephalus (Boophilus) microplus triosephosphate isomerase and BME26 cell growth by monoclonal antibodies. Int J Mol Sci 13:13118–13133
Schadich E, Nylén S, Gurská S, Kotulová J, Andronati S, Pavlovsky V, Soboleva S, Polishchuk P, Hajdúch M, Džubák P (2022) Activity of 1-aryl-4-(naphthalimidoalkyl) piperazine derivatives against Leishmania major and Leishmania mexicana. Parasitol Int 91:102647
Schorderet-Weber S, Noack S, Selzer PM, Kaminsky R (2018) Blocking transmission of vector-borne diseases. Ectoparasites 43–94
Seelig J, Heller RA, Haubruck P, Sun Q, Klingenberg GJ, Hackler J, Crowell HL, Daniel V, Moghaddam A, Schomburg L, Biglari B (2021) Selenium-Binding Protein 1 (SELENBP1) as biomarker for adverse clinical outcome after traumatic spinal cord injury. Front Neurosci 15:680240
Seixas A, dos Santos P, Velloso FF, Vaz IDS, Masuda A, Horn F, Termignoni C (2003) A Boophilus microplus vitellin-degrading cysteine endopeptidase. Parasitology 126:155–163
Seixas A, Estrela AB, Ceolato JC, Pontes EG, Lara F, Gondim KC, Termignoni C (2010) Localization and function of Rhipicephalus (Boophilus) microplus vitellin-degrading cysteine endopeptidase. Parasitology 137:1819–1831
Silva FD, Rossi DCP, Martinez LR, Frases S, Fonseca FL, Campos CBL, Rodrigues ML, Nosanchuk JD, Daffre S (2011) Effects of microplusin, a copper-chelating antimicrobial peptide, against Cryptococcus neoformans. FEMS Microbiol Lett 324:64–72
Sojka D, Franta Z, Horn M, Caffrey CR, Mareš M, Kopáček P (2013) New insights into the machinery of blood digestion by ticks. Trends Parasitol 29:276–285
Sojka D, Snebergerova P, Robbertse L (2021) Protease inhibition—an established strategy to combat infectious diseases. Int J Mol Sci 22(11):5762
Spring MD, Cummings JF, Ockenhouse CF, Dutta S, Reidler R, Angov E, Bergmann-Leitner E, Stewart VA, Bittner S, Juompan L (2009) Phase 1/2a study of the malaria vaccine candidate apical membrane antigen-1 (AMA-1) administered in adjuvant system AS01B or AS02A. PLoS ONE 4:e5254
Stowe DF, Camara AK (2009) Mitochondrial reactive oxygen species production in excitable cells: modulators of mitochondrial and cell function. Antioxid Redox Signal 11:1373–1414
Stutzer C, van Zyl WA, Olivier NA, Richards S, Maritz-Olivier C (2013) Gene expression profiling of adult female tissues in feeding Rhipicephalus microplus cattle ticks. Int J Parasitol 43:541–554
Stutzer C, Richards SA, Ferreira M, Baron S, Maritz-Olivier C (2018) Metazoan parasite vaccines: present status and future prospects. Front Cell Infect Microbiol 8:67
Tandle AT, Calvani M, Uranchimeg B, Zahavi D, Melillo G, Libutti SK (2009) Endothelial monocyte activating polypeptide-II modulates endothelial cell responses by degrading hypoxia-inducible factor-1alpha through interaction with PSMA7, a component of the proteasome. Exp Cell Res 315:1850–1859
Thera MA, Doumbo OK, Coulibaly D, Laurens MB, Ouattara A, Kone AK, Guindo AB, Traore K, Traore I, Kouriba B (2011) A field trial to assess a blood-stage malaria vaccine. N Engl J Med 365:1004–1013
Tirloni L, Braz G, Nunes RD, Gandara ACP, Vieira LR, Assumpcao TC, Sabadin GA, da Silva RM, Guizzo MG, Machado JA, Costa EP, Santos D, Gomes HF, Moraes J, dos Santos Mota MB, Mesquita RD, de Souza Leite M, Alvarenga PH, Lara FA, Seixas A, da Fonseca RN, Fogaça AC, Logullo C, Tanaka AS, Daffre S, Oliveira PL, da Silva Vaz I, Ribeiro JMC (2020) A physiologic overview of the organ-specific transcriptome of the cattle tick Rhipicephalus microplus. Sci Rep 10:18296
Tønnesen M, Penzhorn B, Bryson N, Stoltsz W, Masibigiri T (2004) Displacement of Boophilus decoloratus by Boophilus microplus in the Soutpansberg region, Limpopo province, South Africa. Exp Appl Acarol 32:199–208
Vafopoulou X, Steel CGH (2012) Cytoplasmic travels of the ecdysteroid receptor in target cells: pathways for both genomic and non-genomic actions. Front Endocrinol 3:43–43
Van Oosterwijk JG, Wikel SK (2021) Resistance to ticks and the path to anti-tick and transmission blocking vaccines. Vaccines 9:725
Van Rossum AJ, Jefferies JR, Rijsewijk FA, Lacourse EJ, Teesdale-Spittle P, Barrett J, Tait A, Brophy PM (2004) Binding of hematin by a new class of glutathione transferase from the blood-feeding parasitic nematode Haemonchus contortus. Infect Immun 72:2780–2790
Wang M, Wang J (2020) Glucose transporter GLUT1 influences Plasmodium berghei infection in Anopheles stephensi. Parasit Vectors 13:285
Weidemann A, Breyer J, Rehm M, Eckardt K-U, Daniel C, Cicha I, Giehl K, Goppelt-Struebe M (2013) HIF-1α activation results in actin cytoskeleton reorganization and modulation of Rac-1 signaling in endothelial cells. Cell Commun Signal 11:80
Willadsen P (1997) Novel vaccines for ectoparasites. Vet Parasitol 71:209–222
Woods DJ, Williams TM (2007) The challenges of developing novel antiparasitic drugs. Invertebr Neurosci 7:245–250
Xavier MA, Tirloni L, Pinto AFM, Diedrich JK, Yates JR, Mulenga A, Logullo C, Vaz ID, Seixas A, Termignoni C (2018) A proteomic insight into vitellogenesis during tick ovary maturation. Sci Rep 8:4698
Xavier MA, Tirloni L, Torquato R, Tanaka A, Pinto AFM, Diedrich JK, Yates JR, Vaz ID, Seixas A, Termignoni C (2019) Blood anticlotting activity of a Rhipicephalus microplus cathepsin L-like enzyme. Biochimie 163:12–20
Xavier MA, Brust FR, Waldman J, Macedo AJ, Juliano MA, da Silva Vaz I Jr, Termignoni C (2021) Interfering with cholesterol metabolism impairs tick embryo development and turns eggs susceptible to bacterial colonization. Ticks Tick Borne Dis 12:101790
Yang X, Liu J (2022) Effect of 20-hydroxyecdysone on vitellogenesis in the ixodid tick Haemaphysalis longicornis. Int J Acarol 48:20–26
Zanocco-Marani T, Bateman A, Romano G, Valentinis B, He Z-H, Baserga R (1999) Biological activities and signaling pathways of the granulin/epithelin precursor. Can Res 59:5331–5340
Zhan B, Liu S, Perally S, Xue J, Fujiwara R, Brophy P, Xiao S, Liu Y, Feng J, Williamson A (2005) Biochemical characterization and vaccine potential of a heme-binding glutathione transferase from the adult hookworm Ancylostoma caninum. Infect Immun 73:6903–6911
Zhang XF, Raikhel AS (2021) Hormonal regulation of microRNA expression dynamics in the gut of the yellow fever mosquito Aedes aegypti. RNA Biol 18:1682–1691
Zhang Q, Dou W, Taning CNT, Smagghe G, Wang JJ (2021) Regulatory roles of microRNAs in insect pests: prospective targets for insect pest control. Curr Opin Biotechnol 70:158–166
Zhou J, Schmid T, Frank R, Brüne B (2004) PI3K/Akt is required for heat shock proteins to protect hypoxia-inducible factor 1α from pVHL-independent degradation. J Biol Chem 279:13506–13513
Zhu Y, Si J, Harn DA, Xu M, Ren J, Yu C, Liang Y, Yin X, He W, Cao G (2006) Schistosoma japonicum triose-phosphate isomerase plasmid DNA vaccine protects pigs against challenge infection. Parasitology 132:67–71
Zhu J, Chen L, Raikhel AS (2007) Distinct roles of Broad isoforms in regulation of the 20-hydroxyecdysone effector gene, Vitellogenin, in the mosquito Aedes aegypti. Mol Cell Endocrinol 267:97–105
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Open access funding provided by University of Pretoria. This study was supported by the National Research Foundation of South Africa and the Technology and Innovation Agency of South Africa.
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MF, JC and CS performed the experiments. CMO designed the project. MF, NO, CMO and CS did data analysis. CMO, CS and NO drafted and critically evaluated the data and wrote the manuscript.
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Maritz-Olivier, C., Ferreira, M., Olivier, N.A. et al. Mining gene expression data for rational identification of novel drug targets and vaccine candidates against the cattle tick, Rhipicephalus microplus. Exp Appl Acarol 91, 291–317 (2023). https://doi.org/10.1007/s10493-023-00838-8
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DOI: https://doi.org/10.1007/s10493-023-00838-8