Molecular assessment of voltage-gated sodium channel (VGSC) gene mutations in Rhipicephalus microplus from Guangxi, China.

BACKGROUND: Pyrethroid chemicals are one of the main acaricides used against ticks. Resistance to these chemicals has been reported to be associated with mutations in the voltage-gated sodium channel (VGSC) gene of the Rhipicephalus microplus. This study investigates R. microplus resistance to pyrethroids in Guangxi region of China, marking one of the first research efforts in this area. The findings are intended to provide vital baseline for the effective implementation of localized tick control strategies. METHODS: From March to July 2021, 447 R. microplus tick samples were collected from five prefecture-level cities in Guangxi. Allele-specific polymerase chain reaction (AS-PCR) was used to amplify segments C190A and G215T of the domain II S4-5 linker and T2134A of domain III S6 in the VGSC, to detect nucleotide mutations associated with resistance to pyrethroid acaricides. Subsequent analyses were conducted to ascertain the prevalence, types of mutations, and genotypic distributions within the sampled populations. RESULTS: Mutations within VGSC gene were identified across all five studied populations of R. microplus, although the mutation rates remained generally low. Specifically, the most prevalent mutation was C190A, observed in 4.9% of the samples (22/447), followed by G215T at 4.0% (18/447), and T2134A at 1.3% (6/447). The distribution of mutations across three critical sites of the VGSC gene revealed four distinct mutation types: C190A, G215T, C190A + G215T, and T2134A. Notably, the single mutation C190A had the highest mutation frequency, accounting for 4.3%, and the C190A + G215T combination had the lowest, at only 0.7%. The analysis further identified seven genotypic combinations, with the wild-type combination C/C + G/G + T/T predominating at a frequency of 90.4%. Subsequently, the C/A + G/G + T/T combination was observed at a frequency of 4.3%, whereas the C/C + T/T + T/T combination exhibited the lowest frequency (0.2%). Additionally, no instances of simultaneous mutations at all three sites were detected. Geographical differences in mutation types were apparent. Both samples from Hechi to Chongzuo cities exhibited the same three mutation types; however, C190A was the most prevalent in Hechi, while G215T dominated in Chongzuo. In contrast, samples from Beihai to Guilin each exhibited only one mutation type: G215T occurred in 12.5% (4/32) of Beihai samples, and C190A in 7.5% (4/53) of Guilin samples. CONCLUSIONS: These findings underscore the relatively low frequency of VGSC gene mutations in R. microplus associated with pyrethroid resistance in the Guangxi, China. Moreover, the variation in mutation types and genotypic distributions across different locales highlights the need for regionalized strategies in monitoring and managing pyrethroid resistance in tick populations. This molecular surveillance is crucial for informing targeted control measures and mitigating the risk of widespread resistance emergence.

Profile of cattle breed sensitivity to the tick Rhipicephalus microplus.

Among cattle, Bos taurus breeds and their crosses are more sensitive to tick infestations than Bos indicus breeds that are more resistant to infestation and more adaptable to tropical climates. The presence of susceptible individuals in herds and inadequate tick control lead to direct and indirect losses in the meat production chain, in addition to increased mortality due to cattle tick fever. The objective of this study was to describe, compare and rank the sensitivity of different breeds of stabled cattle to the tick Rhipicephalus microplus and to present, as an innovative result, a scale called the Tick Ruler. Secondary data on the number of retrieved engorged females, engorged female ticks' weight, egg mass weight and number of larvae were extracted from research reports of experiments conducted over 18 years with eight breeds to describe and report the sensitivity of the breeds to artificial infestation by R. microplus larvae. For analyses, the recovery rate of engorged female ticks and the percentile of dispersion of individuals in their respective races were calculated, and comparison of these percentiles between races was performed. The ranking of the percentiles resulted in the organization of the breeds by their susceptibility to R. microplus; we call this scale the "Tick Ruler." The ruler is a simple, easy-to-understand tool that can be used by technicians and producers to evaluate the tick sensitivity of a breed of interest and can assist producers in decision-making to find a balance between increased production gains and the risk of economic losses depending on the breed composition in a cattle herd.

Using QUASR-PCR as a field-based genotyping assay for a tick acaricide resistance marker.

A novel, turnkey, field-based workflow was developed and validated using Rhipicephalus microplus DNA as a template to detect the presence of the voltage-gated sodium channel kdr mutation. The field-based compatible workflow comprises manual sample homogenization for DNA extraction, PCR amplification of the targets in a closed tube, and end-point detection of the PCR products. An R. microplus species-specific assay was also included to confirm species identity and ensure the validity of the kdr mutation assay. The assays were sensitive and specific to the targets, and the workflow resulted in a turnaround time of approximately 1 h at a low cost. The novel combination of PCR with closed-tube and end-point fluorescent detection allows for easy conversion of existing conventional lab-based PCR assays into field-based detection assays. The incorporation of custom-designed 3D-printed components in the workflow provides easy adaptability and modification of the components for diverse nucleic acid detection workflows.

A Novel Receptor Binding Progesterone, a Possible Transregulation Mechanism in the Rhipicephalus microplus-Host Interaction.

BACKGROUND: Hormone receptors exert their function through binding with their ligands, which results in cellular signaling activation mediated by genomic or non-genomic mechanisms. The intrinsic molecular communication of tick Rhipicephalus microplus and its host Bos taurus comprises an endocrine regulation involving hormones. In the present study, we performed a molecular and in silico analysis of a Membrane Associated Progesterone Receptor in R. microplus (RmMAPRC). METHODS: The RmMAPRC protein sequence was analyzed with bioinformatics tools, and its structure was characterized by three-dimensional (3D) modeling and molecular docking. A semi-quantitative reverse transcription and polymerase chain reaction (sqRT-PCR) assessed the RmMAPRC gene presence and relative expression in tick organs and embryonic cells. RESULTS: RmMAPRC relative expression in salivary glands, ovaries, and embryonic cells showed overexpression of 3%, 13%, and 24%, respectively. Bioinformatic analysis revealed that RmMAPRC corresponded to a Progesterone Receptor Membrane Component 1 (RmPGRMC1) of ~23.7 kDa, with an N-terminal transmembrane domain and a C-terminal Cytochrome b5-like heme/steroid binding domain. The docking results suggest that RmPGRMC1 could bind to progesterone (P4), some progestins, and P4 antagonists. The phylogenetic reconstruction showed that Rhipicephalus spp. MAPRC receptors were clustered in a clade that includes R. appendiculatus, R. sanguineus, and R. microplus (RmMAPRC), and mammals and helminths MAPRC receptors clustered in two separated clades away from ticks. CONCLUSIONS: The presence of RmPGRMC1 highlights the importance of transregulation as a conserved adaptive mechanism that has succeeded for arthropod parasites, making it a target for tick control.

A fluorescently-tagged tick kinin neuropeptide triggers peristalsis and labels tick midgut muscles.

Ticks are blood-feeding arthropods that require heme for their successful reproduction. During feeding they also acquire pathogens that are subsequently transmitted to humans, wildlife and/or livestock. Understanding the regulation of tick midgut is important for blood meal digestion, heme and nutrient absorption processes and for aspects of pathogen biology in the host. We previously demonstrated the activity of tick kinins on the cognate G protein-coupled receptor. Herein we uncovered the physiological role of the kinin receptor in the tick midgut. A fluorescently-labeled kinin peptide with the endogenous kinin 8 sequence (TMR-RK8), identical in the ticks Rhipicephalus microplus and R. sanguineus, activated and labeled the recombinant R. microplus receptor expressed in CHO-K1 cells. When applied to the live midgut the TMR-RK8 labeled the kinin receptor in muscles while the labeled peptide with the scrambled-sequence of kinin 8 (TMR-Scrambled) did not. The unlabeled kinin 8 peptide competed TMR-RK8, decreasing confocal microscopy signal intensity, indicating TMR-RK8 specificity to muscles. TMR-RK8 was active, inducing significant midgut peristalsis that was video-recorded and evaluated with video tracking software. The TMR-Scrambled peptide used as a negative control did not elicit peristalsis. The myotropic function of kinins in eliciting tick midgut peristalsis was established.

The TCTP is essential for ovarian development and oviposition of Rhipicephalus haemaphysaloides.

Tick infestations transmit various infectious agents and result in significant socioeconomic consequences. Currently, the primary focus of tick control efforts is identifying potential targets for immune intervention. In a previous study, we identified a highly conserved protein abundant in tick haemolymph extracellular vesicles (EVs) known as translationally controlled tumour protein (TCTP). We have found that native TCTP is present in various tissues of the Rhipicephalus haemaphysaloides tick, including salivary glands, midgut, ovary, and fat body. Notably, TCTP is particularly abundant in the tick ovary and its levels increase progressively from the blood-feeding stage to engorgement. When the TCTP gene was knocked down by RNAi, there was a noticeable delay in ovarian development, and the reproductive performance, in terms of egg quantity and survival, was also hindered. Our investigations have revealed that the observed effects in ovary and eggs in dsRNA-treated ticks are not attributable to cell death mechanisms like apoptosis and autophagy but rather to the reduction in the expression of vitellogenin (Vg1, Vg2, and Vg3) and ferritin (ferritin 1 and ferritin 2) proteins crucial for ovarian development and embryo survival in ticks. Additionally, phylogenetic analysis and structural comparisons of RhTCTP and its orthologues across various tick species, vertebrate hosts, and humans have shown that TCTP is conserved in ticks but differs significantly between ticks and their hosts, particularly in the TCTP_1 and TCTP_2 domains. Overall, TCTP plays a vital role in tick reproductive development and presents itself as a potential target for tick control in both humans and animals.

The complete mitochondrial genome of cattle tick clade C reveals the genetic relationship within Rhipicephalus microplus complex.

Cattle ticks (Rhipicephalus microplus) are important economic ectoparasites causing direct and indirect damage to cattle and leading to severe economic losses in cattle husbandry. It is common knowledge that R. microplus is a species complex including five clades; however, the relationships within the R. microplus complex remain unresolved. In the present study, we assembled the complete mitochondrial genome of clade C by next-generation sequencing and proved its correctness based on long PCR amplification. It was 15,004 bp in length and consisted of 13 protein genes, 22 transfer genes, and two ribosomal genes located in the two strains. There were two copies of the repeat region (pseudo-nad1 and tRNA-Glu). Data revealed that cox1, cox2, and cox3 genes were conserved within R. microplus with small genetic differences. Ka/Ks ratios suggested that 12 protein genes (excluding nad6) may be neutral selection. The genetic and phylogenetic analyses indicated that clade C was greatly close to clade B. Findings in the current study provided more data for the identification and differentiation of the R. microplus complex and made up for the lack of information about R. microplus clade C.

Genetic factors underlying host resistance to Rhipicephalus microplus tick infestation in Braford cattle: a systems biology perspective.

Approximately 80% of the world's cattle are raised in regions with a high risk of tick-borne diseases, resulting in significant economic losses due to parasitism by Rhipicephalus (Boophilus) microplus. However, the lack of a systemic biology approach hampers a comprehensive understanding of tick-host interactions that mediate tick resistance phenotypes. Here, we conducted a genome-wide association study (GWAS) of 2933 Braford cattle and found 340 single-nucleotide polymorphisms (SNPs) associated with tick counts. Gene expression analyses were performed on skin samples obtained from previously tick-exposed heifers with extremely high or low estimated breeding values for R. microplus counts. Evaluations were performed both before and after artificial infestation with ticks. Differentially expressed genes were found within 1-Mb windows centered at significant SNPs from GWAS. A total of 330 genes were related to the breakdown of homeostasis that was induced by larval attachment to bovine skin. Enrichment analysis pointed to a key role of proteolysis and signal transduction via JAK/STAT, NFKB and WNT/beta catenin signaling pathways. Integrative analysis on matrixEQTL revealed two cis-eQTLs and four significant SNPs in the genes peptidyl arginine deiminase type IV (PADI4) and LOC11449251. The integration of genomic data from QTL maps and transcriptome analyses has identified a set of twelve key genes that show significant associations with tick loads. These genes could be key candidates to improve the accuracy of genomic predictions for tick resistance in Braford cattle.

Evaluation of the biological function of ribosomal protein S18 from cattle tick Rhipicephalus microplus.

Rhipicephalus (Boophilus) microplus, also known as the cattle tick, causes severe parasitism and transmits different pathogens to vertebrate hosts, leading to massive economic losses. In the present study, we performed a functional characterization of a ribosomal protein from R. microplus to investigate its importance in blood feeding, egg production and viability. Ribosomal protein S18 (RPS18) is part of the 40S subunit, associated with 18S rRNA, and has been previously pointed to have a secondary role in different organisms. Rhipicephalus microplus RPS18 (RmRPS18) gene expression levels were modulated in female salivary glands during blood feeding. Moreover, mRNA levels in this tissue were 10 times higher than those in the midgut of fully engorged female ticks. Additionally, recombinant RmRPS18 was recognized by IgG antibodies from sera of cattle naturally or experimentally infested with ticks. RNAi-mediated knockdown of the RmRPS18 gene was performed in fully engorged females, leading to a significant (29 %) decrease in egg production. Additionally, egg hatching was completely impaired, suggesting that no viable eggs were produced by the RmRPS18-silenced group. Furthermore, antimicrobial assays revealed inhibitory activities against gram-negative Escherichia coli and gram-positive Staphylococcus aureus bacteria, affecting bacterial growth. Data presented here show the important role of RmRPS18 in tick physiology and suggest that RmRPS18 can be a potential target for the development of novel strategies for tick control.

First detection of Ehrlichia chaffeensis, Ehrlichia canis, and Anaplasma ovis in Rhipicephalus bursa ticks collected from sheep, Turkey.

Anaplasmosis and ehrlichiosis are important tick-borne rickettsial diseases of medical and veterinary importance that cause economic losses in livestock. In this study, the prevalence of Anaplasma ovis, Ehrlichia canis and Ehrlichia chaffeensis was investigated in ticks collected from sheep in various farms in Van province, which is located in the Eastern Anatolian Region of Turkey. The ticks used in this study were collected by random sampling in 26 family farm business in 13 districts of Van province. A total of 688 ticks were collected from 88 sheep and 88 tick pools were created. All ticks identified morphologically as Rhipicephalus bursa. Phylogenetic analysis of Chaperonin and 16S rRNA gene sequences confirmed A. ovis, E. canis and E. chaffeensis in this study. Of the 88 tick pools tested, 28.41% (25/88) were positive for at least one pathogen. Anaplasma DNA was detected in five of the 88 pools (5.68%), E. canis DNA was detected in 19 of the 88 pools (21.59%), and E. chaffeensis DNA was detected in one of the 88 pools (1.14%) of R. bursa ticks. To our knowledge, this is the first report describing the presence of A. ovis, E. canis, and E. chaffeensis in R. bursa ticks collected from sheep in Turkey. Further studies are needed to investigate other co-infections in sheep in Turkey.

Virome analyses of Amblyomma cajennense and Rhipicephalus microplus ticks collected in Colombia.

Tick-borne viruses (TBV) have gained public health relevance in recent years due to the recognition of human-associated fatal cases and the increase in tick-borne disease and transmission. However, many tick species have not been studied for their potential to transmit pathogenic viruses, especially those found in Latin America. To gain better understanding of the tick virome, we conducted targeted amplification using broadly-reactive consensus-degenerate pan-viral targeting viruses from the genera Flavivirus, Bandavirus, Uukuvirus, and Orthonairovirus genus. Additionally, we conducted unbiased metagenomic analyses to investigate the presence of viral RNA sequences in Amblyomma cajennense, A. patinoi and Rhipicephalus microplus ticks collected from a horse slaughter plant in Medellín, Colombia. While no viral products were detected by PCR, results of the metagenomic analyses revealed the presence of viral genomes belonging to the genera Phlebovirus, Bandavirus, and Uukuvirus, including Lihan Tick Virus (LTV), which was previously reported in Rhipicephalus microplus from Colombia. Overall, the results emphasized the enormous utility of the next-generation sequencing in identifying virus genetic diversity presents in ticks and other species of vectors and reservoirs.

Molecular identification of cattle ticks in the Greater Accra Region of Ghana: a high occurrence of Rhipicephalus microplus.

Ticks are competent vectors of a wide range of pathogens. They are of veterinary and public health importance as they affect both animal and human health. Transhumance and the transboundary movements of cattle within the West African Sub-region have facilitated the spread of ticks which threatens the introduction of invasive species. Currently, Rhipicephalus microplus have been identified in the Upper East Region of Ghana which could mean a wider distribution of the species across the country due to livestock trade. This study focused on three sites in the Greater Accra Region, which serves as the gateway to receiving most of the cattle transported from the northern regions of Ghana. Ticks were sampled from August 2022 in the wet season to January 2023 in the dry season. Three tick genera were identified: Amblyomma (19.5%), Hyalomma (1.1%), and Rhipicephalus (79.3%) from the 1,489 feeding ticks collected from cattle. Furthermore, Rhipicephalus microplus, Hyalomma rufipes and Amblyomma variegatum were identified molecularly using primers that target the mitochondrial COI gene. There was a significant association between the tick species and seasons (p < 0.001). Finding R. microplus in this study indicates the extent of the spread of this invasive tick species in Ghana and highlights the need for efficient surveillance systems and control measures within the country.

Relationship between acaricide resistance and acetylcholinesterase gene polymorphisms in the cattle tick Rhipicephalus microplus.

In this study, we aimed to develop a comprehensive methodology for identifying amino acid polymorphisms in acetylcholinesterase transcript 2 (AChE2) in acaricide-resistant Rhipicephalus microplus ticks. This included assessing AChE2 expression levels through qPCR and conducting 3D modeling to evaluate the interaction between acaricides and AChE2 using docking techniques. The study produced significant results, demonstrating that acaricide-resistant R. microplus ticks exhibit significantly higher levels of AChE expression than susceptible reference ticks. In terms of amino acid sequence, we identified 9 radical amino acid substitutions in AChE2 from acaricide-resistant ticks, when compared to the gene sequence of the susceptible reference strain. To further understand the implications of these substitutions, we utilized 3D acaricide-AChE2 docking modeling to examine the interaction between the acaricide and the AChE2 catalytic site. Our models suggest that these amino acid polymorphisms alter the configuration of the binding pocket, thereby contributing to differences in acaricide interactions and ultimately providing insights into the acaricide-resistance phenomenon in R. microplus.

Title: Relations entre la résistance aux acaricides et les polymorphismes du gène de l'acétylcholinestérase chez la tique du bétail Rhipicephalus microplus. Abstract: Notre étude vise à développer une méthodologie complète pour identifier les polymorphismes d'acides aminés dans le transcrit 2 de l'acétylcholinestérase (AChE2) chez les tiques Rhipicephalus microplus résistantes aux acaricides. Cela comprend l'évaluation des niveaux d'expression d'AChE2 via qPCR et la réalisation d'une modélisation 3D pour évaluer l'interaction entre les acaricides et l'AChE2 à l'aide de techniques d'amarrage moléculaire. L'étude a produit des résultats significatifs, démontrant que les tiques R. microplus résistantes aux acaricides présentent des niveaux d'expression d'AChE significativement plus élevés que les tiques sensibles de référence. En termes de séquence d'acides aminés, nous avons identifié 9 substitutions d'acides aminés dans AChE2 provenant de tiques résistantes aux acaricides par rapport à la séquence génétique de la souche sensible de référence. Pour mieux comprendre les implications de ces substitutions, nous avons utilisé la modélisation de l'amarrage acaricide-AChE2 pour examiner l'interaction entre l'acaricide et le site catalytique AChE2. Nos modèles suggèrent que ces polymorphismes d'acides aminés modifient la configuration de la poche de liaison, contribuant ainsi aux différences dans les interactions acaricides et fournissant finalement un aperçu du phénomène de résistance aux acaricides chez R. microplus.

Molecular and next-generation sequencing analysis of tick-borne pathogens of Rhipicephalus ticks (Acari: Ixodidae) in cattle and dogs.

Ticks are small and adaptable arachnid ectoparasites and global carriers of various pathogens that threaten both human and animal health. They are present in many parts of China. A total of 858 ticks were collected from various regions and hosts, then subjected to species identification based on morphological and molecular characteristics, as described in the authors' previous study. Eighty-three individual tick samples were selected for screening pathogens based on metagenomic next-generation sequencing (mNGS) and polymerase chain reaction (PCR) assays. The genomic DNA of tick species was extracted, and amplification of the bacterial 16S rRNA gene was carried out from DNA of individual ticks using V3-V4 hypervariable regions, before subjecting to metagenomic analysis. Each tick underwent specific PCR tests for identifying the bacterial species present, including Anaplasma, Ehrlichia, Coxiella, and Rickettsia, and also protozoans such as Babesia, Theileria, and Hepatozoon. Illumina NovaSeq sequencing results revealed that the dominant phylum and family in Rhipicephalus spp. were Bacteroidota and Muribaculaceae, respectively. Alpha diversity patterns varied depending on tick sex (R. linnaei only), species and location, but not on host. Furthermore, bacterial pathogens, including A. marginale (58 %, 29/50), A. platys (6 %, 3/50), E. minasensis (2 %, 1/50), Ehrlichia sp. (10 %, 5/50), T. sinensis (24 %, 12/50), T. orientalis (54 %, 27/50) and Coxiella-like bacteria (CLB) (80 %, 40/50) were detected in R. microplus, while E. canis (33.33 %, 10/30), H. canis (20 %, 6/30) and CLB (100 %, 30/30) were detected in R. linnaei. Also, Anaplasma sp. (33.33 %, 1/3), A. marginale (33.33 %, 1/3), R. felis (33.33 %, 1/3) and CLB (100 %, 3/3) were detected in R. haemaphysaloides. Dual and triple co-infections involving pathogens or CLB were detected in 84.00 % of R. microplus, 66.66 % of R. haemaphysaloides, and 33.00 % of R. linnaei. The report on microbial communities and pathogens, which found from Rhipicephalus spp. in Hainan Island, is an important step towards a better understanding of tick-borne disease transmission. This is the first report in the area on the presence of Anaplasma sp., A. marginale, R. felis and Coxiella, in R. haemaphysaloides.

Effect of an altitudinal gradient on the morphology, molecular identification and distribution of Rhipicephalus linnaei in Veracruz, Mexico.

Studies of morphological and genetic variation in vector populations across environmental gradients can help researchers to estimate species' responses to climate change scenarios and the potential risk of disease-causing pathogen expansion, which impacts negatively on human health. In this study, we analysed the effect of altitudinal gradients on the phenotypic response of the hard tick of medical and veterinary importance, Rhipicephalus sanguineus sensu lato (s.l.). Specimens of R. sanguineus s.l. were collected from host animals in one of Mexico's regions with high climatic heterogeneity (Veracruz), and geometric morphometric theory was employed to assess the response of three morphological characters to the altitudinal gradient. Additionally, genetic similarity data were provided, and ecological niche models were used to project the climatic distribution in the region. Our results demonstrate that the shape and size of ticks respond to altitude. Molecular identification indicate that all analysed samples correspond to the tropical lineage recently named Rhipicephalus linnaei. According to ecological niche models, the mean annual temperature contributes significantly to the spatial distribution of this tick species, with areas of higher suitability in the mountainous region. These changes in morphological structure and the presence of ticks at higher altitudinal gradients suggest that R. linnaei has a high potential for adaptation. Due to the variability of ecosystems in the state of Veracruz, our results could be valuable in assessing the response of this tick in a changing environment, aiding in predicting future scenarios in the distribution and abundance of this species.

Expression network analysis of bovine skin infested with Rhipicephalus australis identifies pro-inflammatory genes contributing to tick susceptibility.

The skin is the primary feeding site of ticks that infest livestock animals such as cattle. The highly specialised functions of skin at the molecular level may be a factor contributing to variation in susceptibility to tick infestation; but these remain to be well defined. The aim of this study was to investigate the bovine skin transcriptomic profiles of tick-naïve and tick-infested cattle and to uncover the gene expression networks that influence contrasting phenotypes of host resistance to ticks. RNA-Seq data was obtained from skin of Brangus cattle with high (n = 5) and low (n = 6) host resistance at 0 and 12 weeks following artificial tick challenge with Rhipicephalus australis larvae. No differentially expressed genes were detected pre-infestation between high and low resistance groups, but at 12-weeks there were 229 differentially expressed genes (DEGs; FDR < 0.05), of which 212 were the target of at least 1866 transcription factors (TFs) expressed in skin. Regulatory impact factor (RIF) analysis identified 158 significant TFs (P < 0.05) of which GRHL3, and DTX1 were also DEGs in the experiment. Gene term enrichment showed the significant TFs and DEGs were enriched in processes related to immune response and biological pathways related to host response to infectious diseases. Interferon Type 1-stimulated genes, including MX2, ISG15, MX1, OAS2 were upregulated in low host resistance steers after repeated tick challenge, suggesting dysregulated wound healing and chronic inflammatory skin processes contributing to host susceptibility to ticks. The present study provides an assessment of the bovine skin transcriptome before and after repeated tick challenge and shows that the up-regulation of pro-inflammatory genes is a prominent feature in the skin of tick-susceptible animals. In addition, the identification of transcription factors with high regulatory impact provides insights into the potentially meaningful gene-gene interactions involved in the variation of phenotypes of bovine host resistance to ticks.

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.

Phenotypic and genotypic characterization of acaricide resistance in Rhipicephalus microplus field isolates from South Africa and Brazil.

Rhipicephalus (Boophilus) microplus is one of the most successful ticks infesting cattle around the world. This highly-invasive species transmits cattle parasites that cause cattle fever leading to a high socio-economic burden. Tick eradication programs have often failed, due to the development of acaricide resistance. Here we characterize acaricide resistance in a large number of tick isolates from regions in South Africa (KwaZulu Natal, Mpumalanga, Western & Eastern Cape provinces) and two Brazilian regions. By means of Larval Packet Tests (LPT's) acaricide resistance was evaluated against five commonly used acaricides (chlorfenvinphos, fipronil, deltamethrin, amitraz, and ivermectin). Furthermore, the coding region containing the knock down resistance (kdr) mutation, known to result in pyrethroid resistance, was sequenced. Resistance to at least one acaricide class was reported in each of the five regions, and a high proportion of tick isolates exhibited multi-resistance to at least two acaricide classes (range: 22.2-80.0%). Furthermore, resistance ratios (RR) showed high spatial variation (intercontinental, as well as regional) but low regional spatial autocorrelation. Previous and current acaricide use correlated with current RR, and several combinations of acaricide RR were positively correlated. Moreover, fipronil resistance tended to be higher in farms with more intense acaricide use. The kdr-mutations provided the ticks a fitness advantage under the selection pressure of synthetic pyrethroids based on population (kdr-allele frequency) and individual level data (genotypes). The data show the threat of acaricide (multi-)resistance is high in Brazil and South Africa, but acaricide specific levels need to be assessed locally. For this purpose, gathering complementary molecular information on mutations that underlie resistance can reduce costs and expedite necessary actions. In an era of human-caused habitat alterations, implementing molecular data-driven programs becomes essential in overcoming tick-induced socio-economic losses.

Comparative analysis of Presence-Absence gene Variations in five hard tick species: impact and functional considerations.

Tick species are vectors of harmful human and animal diseases, and their expansion is raising concerns under the global environmental changes' scenario. Ticks host and transmit bacteria, protozoa and viruses, making the understanding of host-pathogen molecular pathways critical to development of effective disease control strategies. Despite the considerable sizes and repeat contents of tick genomes, individual tick genomics is perhaps the most effective approach to reveal genotypic traits of interest. Presence-Absence gene Variations (PAVs) can contribute to individual differences within species, with dispensable genes carried by subsets of individuals possibly underpinning functional significance at individual or population-levels. We exploited 350 resequencing datasets of Dermacentor silvarum, Haemaphysalis longicornis, Ixodes persulcatus, Rhipicephalus microplus and Rhipicephalus sanguineus hard tick specimens to reveal the extension of PAV and the conservation of dispensable genes among individuals and, comparatively, between species. Overall, we traced 550-3,346 dispensable genes per species and were able to reconstruct 5.3-7 Mb of genomic regions not included in the respective reference genomes, as part of the tick pangenomes. Both dispensable genes and de novo predicted genes indicated that PAVs preferentially impacted mobile genetic elements in these 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.