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.

Towards modelling tick-virus interactions using the weakly pathogenic Sindbis virus: Evidence that ticks are competent vectors.

Most tick-borne viruses (TBVs) are highly pathogenic and require high biosecurity, which severely limits their study. We found that Sindbis virus (SINV), predominantly transmitted by mosquitoes, can replicate in ticks and be subsequently transmitted, with the potential to serve as a model for studying tick-virus interactions. We found that both larval and nymphal stages of Rhipicephalus haemaphysaloides can be infected with SINV-wild-type (WT) when feeding on infected mice. SINV replicated in two species of ticks (R. haemaphysaloides and Hyalomma asiaticum) after infecting them by microinjection. Injection of ticks with SINV expressing enhanced Green Fluorescent Protein (eGFP) revealed that SINV-eGFP specifically aggregated in the tick midguts for replication. During blood-feeding, SINV-eGFP migrated from the midguts to the salivary glands and was transmitted to a new host. SINV infection caused changes in expression levels of tick genes related to immune responses, substance transport and metabolism, cell growth and death. SINV mainly induced autophagy during the early stage of infection; with increasing time of infection, the level of autophagy decreased, while the level of apoptosis increased. During the early stages of infection, the transcript levels of immune-related genes were significantly upregulated, and then decreased. In addition, SINV induced changes in the transcription levels of some functional genes that play important roles in the interactions between ticks and tick-borne pathogens. These results confirm that the SINV-based transmission model between ticks, viruses, and mammals can be widely used to unravel the interactions between ticks and viruses.

Epidemiological Investigation of Yak (Bos grunniens) Fascioliasis in the Pastoral Area of Qinghai-Tibet Plateau, China.

The objective of this investigation was to understand the epidemiology of fascioliasis in yaks in the alpine pastoral areas of the Qinghai-Tibet Plateau, China. The prevalence of Fasciola hepatica infection was estimated by examining eggs in the feces of yaks and by autopsy after the slaughter. Yaks were sampled from a total of 16 representative counties in Qinghai province, and risk factors were assessed based on regional and age characteristics. Fecal samples were obtained from 1542 yaks aged 0-1 (<1 year old), 1-2 (≥1 year old and <3 years old), and over 3 years (≥3 years old). In addition, 242 yaks over 3 years old who had not undergone fecal examinations were randomly selected for autopsy. A total of 267 fecal samples were positive for Fasciola spp. eggs. The average infection rate was 17.32% (0-60.61%), and the average infection intensity was 51.9 eggs per gram (epg) of feces, with intensities ranging from 18 to 112 epg. In Maduo, Dari, Zhiduo, Chengduo, and Datong counties, the Fasciola spp. eggs infection rate was zero. Fasciola spp. adult flukes were detected in 66 out of 242 yaks at autopsy, with a total infection rate of 27.27% and an average infection intensity of 21.2 (adult worms), with intensities ranging from 3 to 46 worms. Logistic regression model analysis showed that age was a significant risk factor for yak infection with Fasciola spp. In addition, the risk varied between regions: Haiyan, Gangcha, Duran, and Wulan were all high-risk areas for yak infection with Fasciola spp. The spatial distribution of the Fasciola spp. infection rate in each region showed a very weak negative correlation (Moran's I = -0.062), Duran formed a spatial distribution of high-low clusters with surrounding areas, and Datong formed a low-high clustering distribution characteristic with the surrounding areas. This investigation revealed that the infection rate of Fasciola spp. in yaks was higher on the Qinghai-Tibet Plateau. Increasing age was a risk factor for infection with Fasciola spp.; different regions also have a different risk of Fasciola spp. infection. Only two regions showed clustering characteristics in the spatial distribution of infection rates. These findings extend the epidemiological information on Fasciola spp. infection in yaks and provide baseline data for the execution of control measures against Fasciola spp. infection.

Repellent effects of Chinese cinnamon oil on nymphal ticks of Haemaphysalis longicornis, Rhipicephalus haemaphysaloides, and Hyalomma asiaticum.

The repellent activity of Chinese cinnamon oil (Cinnamomum cassia) on nymphal ticks (Haemaphysalis longicornis Neumann, Rhipicephalus haemaphysaloides Supino, and Hyalomma asiaticum Schulze and Schlottke) was evaluated in a sample Y-tube bioassay. The results were based on the vertical migration of ticks during the host-seek phase and showed a dose-dependent repellent effect of Chinese cinnamon oil on the tested nymphs after 6 h. For H. longicornis, R. haemaphysaloides, and H. asiaticum at the concentrations (vol/vol) of 3, 3, and 1.5%, the repellent percentages over time were 68-97, 69-94, and 69-93%, respectively, which indicated strong repellent activities against ticks, similar to the positive control DEET (N,N-diethyl-3-methylbenzamide). Chinese cinnamon oil exerted the strongest effect on H. asiaticum nymphs. To our knowledge, this is the first study to investigate the repellent effects of Chinese cinnamon oil on ticks. Chinese cinnamon oil has considerable potential and should be developed as a practical tick repellent.

Proteomic analysis of extracellular vesicles from tick hemolymph and uptake of extracellular vesicles by salivary glands and ovary cells.

BACKGROUND: Extracellular vesicles (EVs) are a heterogeneous group of cell-derived membranous structures that are important mediators of intercellular communication. Arthropods transport nutrients, signaling molecules, waste and immune factors to all areas of the body via the hemolymph. Little is known about tick hemolymph EVs. METHODS: Hemolymph was collected from partially fed Rhipicephalus haemaphysaloides and Hyalomma asiaticum ticks by making an incision with a sterile scalpel in the middle (between the femur and metatarsus) of the first pair of legs, which is known as leg amputation. EVs were isolated from hemolymph by differential centrifugation and characterized by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Proteins extracted from the hemolymph EVs were analyzed by 4D label-free proteomics. The EVs were also examined by western blot and immuno-electron microscopy analysis. Intracellular incorporation of PHK26-labeled EVs was tested by adding labeled EVs to tick salivary glands and ovaries, followed by fluorescence microscopy. RESULTS: In this study, 149 and 273 proteins were identified by 4D label-free proteomics in R. haemaphysaloides and H. asiaticum hemolymph EVs, respectively. TEM and NTA revealed that the sizes of the hemolymph EVs from R. haemaphysaloides and H. asiaticum were 133 and 138 nm, respectively. Kyoto Encyclopedia of Genes and Genomes and Gene Ontology enrichment analyses of identified proteins revealed pathways related to binding, catalytic and transporter activity, translation, transport and catabolism, signal transduction and cellular community. The key EV marker proteins RhCD9, RhTSG101, Rh14-3-3 and RhGAPDH were identified using proteomics and western blot. The presence of RhFerritin-2 in tick hemolymph EVs was confirmed by western blot and immuno-electron microscopy. We demonstrated that PKH26-labeled hemolymph EVs are internalized by tick salivary glands and ovary cells in vitro. CONCLUSIONS: The results suggest that tick EVs are secreted into, and circulated by, the hemolymph. EVs may play roles in the regulation of tick development, metabolism and reproduction.

Current Knowledge on Chemosensory-Related Candidate Molecules Potentially Involved in Tick Olfaction via Haller's Organ.

Ticks are obligatory hematophagous ectoparasites and vectors of many animal and human pathogens. Chemosensation plays a significant role in tick communication with their environment, including seeking out blood meal hosts. Studies on the structure and function of Haller's organ and its components have improved our understanding regarding tick olfaction and its chemical ecology. Compared with the knowledge on insect olfaction, less is known about the molecular basis of olfaction in ticks. This review focused on the chemosensory-related candidate molecules likely involved in tick olfaction. Members of the ionotropic receptor family and a new class of odorant-binding proteins are now known to be involved in tick olfaction, which appear to differ from that of insects. These candidate molecules are more closely related to those of mites and spiders than to other arthropods. The amino acid sequences of candidate niemann-pick type C2 and microplusin-like proteins in ticks exhibit features indicating their potential role as binding proteins. In the future, more comprehensive pertinent research considering the existing shortcomings will be required to fully understand the molecular basis of tick olfactory chemoreception. This information may contribute to the development of new molecular-based control mechanisms to reduce tick populations and related disease transmission.

Systematic Determination of TCR-Antigen and Peptide-MHC Binding Kinetics among Field Variants of a Theileria parva Polymorphic CTL Epitope.

CTLs are known to contribute to immunity toward Theileria parva, the causative agent of East Coast fever. The Tp967-75 CTL epitope from the Muguga strain of T. parva is polymorphic in other parasite strains. Identifying the amino acids important for MHC class I binding, as well as TCR recognition of epitopes, can allow the strategic selection of Ags to induce cellular immunity toward T. parva In this study, we characterized the amino acids important for MHC class I binding and TCR recognition in the Tp967-75 epitope using alanine scanning and a series of variant peptide sequences to probe these interactions. In a peptide-MHC class I binding assay, we found that the amino acids at positions 1, 2, and 3 were critical for binding to its restricting MHC class I molecule BoLA-1*023:01. With IFN-γ ELISPOT and peptide-MHC class I Tet staining assays on two parasite-specific bovine CTL lines, we showed that amino acids at positions 5-8 in the epitope were required for TCR recognition. Only two of eight naturally occurring polymorphic Tp9 epitopes were recognized by both CTLs. Finally, using a TCR avidity assay, we found that a higher TCR avidity was associated with a stronger functional response toward one of two variants recognized by the CTL. These data add to the growing knowledge on the cross-reactivity of epitope-specific CTLs and specificities that may be required in the selection of Ags in the design of a wide-spectrum vaccine for East Coast fever.

Characterization of a single-domain von Willebrand factor type C protein (HaSVC) from the salivary gland of the tick Hyalomma asiaticum.

As a widely distributed arthropod and vector for various pathogens, Hyalomma asiaticum presents great risk and potential losses in animal husbandry. Effective measures, including the use of vaccines, are necessary for controlling ticks and tick-borne diseases. A concise understanding of the tick-host interaction associated molecules and pathways is required for vaccine development. In the present study, a protein containing a single-domain von Willebrand factor type C (HaSVC) was isolated from H. asiaticum and was subjected to functional identification. As a result, the full-length sequence of the HaSVC (506 bp) gene was obtained, which putatively encodes 100 amino acids with a predicted molecular mass of 11 kDa, excluding the 23-amino acid signal peptide. HaSVC contains 8 cysteines to form 4 disulfide bonds. The native HaSVC protein was detected in multiple tick organs. HaSVC neither attenuated the anti-coagulation process nor directly affected the blood feeding of adult ticks. However, the purified recombinant protein HaSVC (rHaSVC/GST) significantly increased the proliferation of mice spleen cells. This might suggest a regulatory function for HaSVC on inflammation, thus providing new information that may explain the "crosstalk" between ticks and hosts.

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

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

Corrigendum: The Tick Microbiota Dysbiosis Promote Tick-Borne Pathogen Transstadial Transmission in a Babesia microti Infected Mouse Model.

[This corrects the article DOI: 10.3389/fcimb.2021.713466.].

Identification of the Bcl-2 and Bax homologs from Rhipicephalus haemaphysaloides and their function in the degeneration of tick salivary glands.

BACKGROUND: The salivary glands of female ticks degenerate rapidly by apoptosis and autophagy after feeding. Bcl-2 family proteins play an important role in the apoptosis pathways, but the functions of these proteins in ticks are unclear. We studied Bcl-2 and Bax homologs from Rhipicephalus haemaphysaloides and determined their functions in the degeneration of the salivary glands. METHODS: Two molecules containing conserved BH (Bcl-2 family homology) domains were identified and named RhBcl-2 and RhBax. After protein purification and mouse immunization, specific polyclonal antibodies (PcAb) were created in response to the recombinant proteins. Reverse transcription quantitative PCR (RT-qPCR) and western blot were used to detect the presence of RhBcl-2 and RhBax in ticks. TUNEL assays were used to determine the level of apoptosis in the salivary glands of female ticks at different feeding times after gene silencing. Co-transfection and GST pull-down assays were used to identify interactions between RhBcl-2 and RhBax. RESULTS: The RT-qPCR assay revealed that RhBax gene transcription increased significantly during feeding at all tick developmental stages (engorged larvae, nymphs, and adult females). Transcriptional levels of RhBcl-2 and RhBax increased more significantly in the female salivary glands than in other tissues post engorgement. RhBcl-2 silencing significantly inhibited tick feeding. In contrast, RhBax interference had no effect on tick feeding. TUNEL staining showed that apoptosis levels were significantly reduced after interference with RhBcl-2 expression. Co-transfection and GST pull-down assays showed that RhBcl-2 and RhBax could interact but not combine in the absence of the BH3 domain. CONCLUSIONS: This study identified the roles of RhBcl-2 and RhBax in tick salivary gland degeneration and finds that the BH3 domain is a key factor in their interactions.

The Tick Microbiota Dysbiosis Promote Tick-Borne Pathogen Transstadial Transmission in a Babesia microti-Infected Mouse Model.

Ticks are obligate hematophagous ectoparasites. They are important vectors for many pathogens, of both medical and veterinary importance. Antibiotic residues in animal food are known, but very little is known about the effects of antibiotic residues in animals on the microbiome diversity of ticks and tick-borne pathogen transmission. We used a Haemaphysalis longicornis-infested mouse model to evaluate the effect of antibiotic usage on tick microbiome. Nymphal ticks were fed on an antibiotic cocktail-treated or water control mice. Adult ticks molted from nymphs fed on the antibiotic cocktail-treated mouse had a dysbiosed microbiota. Nymphal ticks were also fed on a B. microti-infected mice that had been treated with antibiotic cocktail or water. We found that the B. microti infection in adult ticks with a dysbiosed microbiota (44.7%) was increased compared with the control adult ticks (24.2%) by using qPCR targeting 18S rRNA gene. This may increase the risk of tick-borne pathogens (TBPs) transmission from adult ticks to a vertebrate host. These results show that an antibiotic-treated mouse can induce tick microbiota dysbiosis. Antibiotic treatment of B. microti-infected mouse poses the possibility of increasing transstadial transmission of B. microti from the nymph to the adult H. longicornis. These findings suggest that B. microti transmission may be exacerbated in high antibiotic usage areas.

Engorgement of Rhipicephalus haemaphysaloides ticks blocked by silencing a protein inhibitor of apoptosis.

Inhibitors of apoptosis (IAPs) are regulators of cell death and may play a role in the salivary glands of ticks during blood-feeding. We cloned the open reading frame (ORF) sequence of the IAP gene in Rhipicephalus haemaphysaloides (RhIAP). The RhIAP ORF of 1887 bp encodes a predicted protein of 607 amino acids, which contains three baculovirus IAP repeat domains and a RING finger motif. A real-time PCR assay showed that RhIAP mRNA was expressed in all the tick developmental stages (eggs, larvae, nymphs, and adults) and in all tissues examined (midgut, ovary, salivary glands, fat body, and hemolymph). Western blot showed that the protein level of RhIAP in salivary glands increased during tick blood-feeding and decreased towards the end of tick engorgement. RhIAP gene silencing in vitro experiments with salivary glands demonstrated that RhIAP could be effectively knocked down within 48 h after dsRNA treatment, and as a consequence, salivary glands displayed apoptotic morphology. RhIAP gene silencing also inhibited tick blood-feeding and decreased the engorgement rate. These data suggest that RhIAP might be a suitable RNAi target for tick control.

Evaluation of enzymatic activity of Babesia microti thioredoxin reductase (Bmi TrxR)-mutants and screening of its potential inhibitors.

Babesia microti is a zoonotic pathogen that mainly parasitizes mammalian erythrocytes. Oxidative stress can induce gene mutation, protein denaturation and lipid peroxidation, such as reactive oxygen species (ROS) induced by hypoxic environment and the host immune system. An antioxidase, B. microti thioredoxin reductase (Bmi TrxR), has been identified in B. microti. We used a combination of homology modeling and domain prediction to explore the functional sites of Bmi TrxR and found that TrxR has three domains. Constructed a mutant pool which His-tag were at the N-terminus (TrxR-Nhis, C105-Nhis, C110-Nhis, C105110-Nhis, C547-Nhis, C552-Nhis, C547552-Nhis) and the His tag were at the N- and C-terminus (TrxR-NChis, C547-NChis, C552-NChis, C547552-NChis). The proteins were expressed as His-tagged fusion proteins in Escherichia coli. The His-tag of TrxR C-terminus were affected the reaction with Trx. The inhibitory efficiency of DNCB was decreased for mutant C547, compared with recombinant TrxR, indicating that the action site of DNCB might be cysteine at position 547. These results indicate that the N-terminal active site of Bmi TrxR plays an important role in accepting electrons and promotes electron transfer. The C-terminus His tag of Bmi TrxR affected the electron transfer and the reducing activity of Bmi TrxR. Reduce reactive oxygen produced in oxidative stress was reduced by Bmi TrxR, which is beneficial to Babesia survival. Therefore, reduction site of TrxR may become a potential target for Babesia microti treatment.

ATG5 is instrumental in the transition from autophagy to apoptosis during the degeneration of tick salivary glands.

Female tick salivary glands undergo rapid degeneration several days post engorgement. This degeneration may be caused by the increased concentration of ecdysone in the hemolymph during the fast feeding period and both autophagy and apoptosis occur. In this work, we first proved autophagy-related gene (ATG) and caspase gene expression peaks during degeneration of the tick salivary glands. We explored the regulatory role of Rhipicephalus haemaphysaloides autophagy-related 5 (RhATG5) in the degeneration of tick salivary glands. During the fast feeding phase, RhATG5 was cleaved and both calcium concentration and the transcription of Rhcalpains increased in the salivary glands. Recombinant RhATG5 was cleaved by µ-calpain only in the presence of calcium; the mutant RhATG5191-199Δ was not cleaved. Treatment with 20-hydroxyecdysone (20E) led to programmed cell death in the salivary glands of unfed ticks in vitro, RhATG8-phosphatidylethanolamine (PE) was upregulated in ticks treated with low concentration of 20E. Conversely, RhATG8-PE decreased and Rhcaspase-7 increased in ticks treated with a high concentration of 20E and transformed autophagy to apoptosis. High concentrations of 20E led to the cleavage of RhATG5. Calcium concentration and expression of Rhcalpains were also upregulated in the tick salivary glands. RNA interference (RNAi) of RhATG5 in vitro inhibited both autophagy and apoptosis of the tick salivary glands. RNAi of RhATG5 in vivo significantly inhibited the normal feeding process. These results demonstrated that high concentrations of 20E led to the cleavage of RhATG5 by increasing the concentration of calcium and stimulated the transition from autophagy to apoptosis.

Proteomic Analysis of Exosome-Like Vesicles Isolated From Saliva of the Tick Haemaphysalis longicornis.

Extracellular vesicles (EVs), are considered as vehicles of cellular communication. Parasites usually release EVs in their excretory-secretory products to modulate host environment. However, little is known about the secretion of EVs by ticks. In this study, we show for the first time that the tick Haemaphysalis longicornis secretes EVs in saliva that resembles exosomes. EVs were purified from pilocarpine induced saliva of partially engorged H. longicornis ticks. Electron microscopy analysis revealed the presence of exosome-like vesicles with a size of 100 nm. Proteomic analysis by LC-MS/MS identified a total of 356 proteins in tick-derived EVs. Proteome data of tick-derived EVs was validated by Western blot analysis. Immunodetection of Hsp70 and GAPDH proteins indicated that the proteomics data of tick-derived EVs were highly reliable. Bioinformatics analysis (Gene Ontology) indicated association of certain biological and molecular functions with proteins which may be helpful during tick development. Likewise, KEGG database revealed involvement of vesicular proteins in proton transport, detoxification, ECM-receptor interaction, ribosome, RNA transport, ABC transporters, and oxidative phosphorylation. The results of this study provide evidence that EVs are being secreted in tick saliva and suggest that tick saliva-derived EVs could play important roles in host-parasite relationships. Moreover, EVs could be a useful tool in development of vaccines or therapeutics against ticks.

miRNA profile of extracellular vesicles isolated from saliva of Haemaphysalis longicornis tick.

Extracellular vesicles (EVs) play a key role in host-parasite interactions. Previous studies have shown that parasites can release microRNA (miRNA) containing EVs, which can transfer their contents to host cells and regulate gene expression in recipient cells. However, a little is known about the secretion of EVs by the ticks. This study was therefore, carried out to examine the saliva of ticks for the presence of miRNA containing EVs. Vesicles were purified from saliva of partially engorged Haemaphysalis longicornis ticks. Transmission electron microscopy (TEM) was carried out to confirm that vesicles within saliva were EVs based on size and morphology. Total RNA was extracted from EVs and was analyzed by deep sequencing to determine miRNA profile. TEM analysis confirmed the presence of extracellular vesicle-like structures within tick saliva. RNA-seq analysis showed that tick-derived EVs contained small non-coding RNA populations including miRNAs. The analysis of tick-derived EVs identified 36 known miRNAs, 34 novel miRNAs and 842 novel Piwi-interacting RNAs (piRNA). The results of this study provide evidence that EVs containing miRNAs can be secreted by the ticks and suggest that vesicles could transfer these miRNAs to modulate host cell functions.

A cysteine protease of Babesia microti and its interaction with tick cystatins.

Babesiosis is a tick-borne protozoonosis caused by Babesia, which can cause fever, hemolytic anemia, hemoglobinuria, and even death. Babesia microti is a parasite found in rodents and can be pathogenic to humans. In this study, the full-length cDNA of a B. microti cysteine protease (BmCYP) was expressed and the recombinant rBmCYP protein analyzed and characterized. BmCYP is encoded by an ORF of 1.3 kb, with a predicted molecular weight of 50 kDa and a theoretical pI of 8.5. The amino acid sequence of BmCYP exhibits an identity of 32.9 to 35.2% with cysteine proteases of Babesia ovis, Babesia bovis, and Theileria, respectively. The results of the proteinase assays show that rBmCYP has cysteine protease enzymatic activity. In addition, we demonstrate that tick cystatins rRhcyst-1 and rRhcyst-2 were able to effectively inhibit the activity of rBmCYP; the inhibition rates were 57.2% and 30.9%, respectively. Tick cystatins Rhcyst-1 and Rhcyst-2 were differentially expressed in ticks that fed on Babesia-infected mice relative to non-infected control ticks. Our results suggest that BmCYP is a functional enzyme with cysteine protease enzymatic activity and may be involved in tick-B. microti interactions.

Initiator and executioner caspases in salivary gland apoptosis of Rhipicephalus haemaphysaloides.

BACKGROUND: Apoptosis is fundamental in maintaining cell balance in multicellular organisms, and caspases play a crucial role in apoptosis pathways. It is reported that apoptosis plays an important role in tick salivary gland degeneration. Several different caspases have been found in ticks, but the interactions between them are currently unknown. Here, we report three new caspases, isolated from the salivary glands of the tick Rhipicephalus haemaphysaloides. METHODS: The full-length cDNA of the RhCaspases 7, 8 and 9 genes were obtained by transcriptome, and RhCaspases 7, 8 and 9 were expressed in E. coli; after protein purification and immunization in mice, specific polyclonal antibodies (PcAb) were created in response to the recombinant protein. Reverse-transcription quantitative PCR (RT-qPCR) and western blot were used to detect the existence of RhCaspases 7, 8 and 9 in ticks. TUNEL assays were used to determine the apoptosis level in salivary glands at different feeding times after gene silencing. The interaction between RhCaspases 7, 8 and 9 were identified by co-transfection assays. RESULTS: The transcription of apoptosis-related genes in R. haemaphysaloides salivary glands increased significantly after tick engorgement. Three caspase-like molecules containing conserved caspase domains were identified and named RhCaspases 7, 8 and 9. RhCaspase8 and RhCaspase9 contain a long pro-domain at their N-terminals. An RT-qPCR assay demonstrated that the transcription of these three caspase genes increased significantly during the engorged periods of the tick developmental stages (engorged larval, nymph, and adult female ticks). Transcriptional levels of RhCaspases 7, 8 and 9 in salivary glands increased more significantly than other tissues post-engorgement. RhCaspase9-RNAi treatment significantly inhibited tick feeding. In contrast, knockdown of RhCaspase7 and RhCaspase8 had no influence on tick feeding. Compared to the control group, apoptosis levels were significantly reduced after interfering with RhCaspase 7, 8 and 9 expressions. Co-transfection assays showed RhCaspase7 was cleaved by RhCaspases 8 and 9, demonstrating that RhCaspases 8 and 9 are initiator caspases and RhCaspase7 is an executioner caspase. CONCLUSIONS: To the best of our knowledge, this is the first study to identify initiator and executioner caspases in ticks, confirm the interaction among them, and associate caspase activation with tick salivary gland degeneration.

A family of serine protease inhibitors (serpins) and its expression profiles in the ovaries of Rhipicephalus haemaphysaloides.

Serpins are evolutionarily conserved serine protease inhibitors found in many organisms. In arthropods, serpins are involved in feeding, development, oviposition, anti-coagulation and innate immune responses. We characterized of 11 serpins in the tick Rhipicephalus haemaphysaloides. These serpins have orthologous genes in other ticks, as indicated by phylogenetic analysis. Analysis of the reactive center loop and hinge regions of the protein sequences indicated that RHS7 encodes proteins that may lack proteinase inhibitor activity. All R. haemaphysaloides serpins had high amino acid sequence identities to Rhipicephalus microplus serpins. Tissue and temporal transcriptional profiling of eight R. haemaphysaloides serpins located in the ovaries demonstrated that they are transcribed during feeding and oviposition. These suggested their participation in the regulation of tick physiology. Immune serum from rabbits repeatedly infested with larvae, nymphs and adults of R. haemaphysaloides can recognize multiple recombinant serpins, respectively. After gene silencing, the blood feeding to repletion time was significantly longer and the 24 h attachment rate was significantly lower in the RHS3 and RHS7 knock down groups. The RHS9 and RHS11 silenced ticks had significant reduction in repletion time and egg-laying rate. Egg hatchability was significantly decreased in RHS4, RHS5 and RHS9 silenced ticks. All groups had significant reductions in engorged body weight. This study increases information on the serpins of R. haemaphysaloides and suggests that some RHSs are potential targets for development of tick vaccines.