Search

The Ornithodoros moubata (Om) soft tick, a vector for diseases like tick‐borne human relapsing fever and African swine fever, poses challenges to conventional control methods. With diminishing insecticide efficacy, harnessing the tick's microbiota through innovative approaches like microbiota‐driven vaccination emerges as a promising strategy for sustainable and targeted disease control. This study investigated the intricate relationship between Pseudomonas, a keystone taxon in the Om microbiome, and its impact on tick fitness, microbiome structure and network dynamics. Utilizing in silico analyses and empirical vaccination experiments, the role of Pseudomonas within microbial networks in the tick midguts (MG) and salivary glands (SG) of Om was studied. Additionally, the consequences of anti‐microbiota vaccines targeting Pseudomonas and Lactobacillus on tick fitness, microbiome diversity and community assembly were explored. The result of the study shows that in Om, Pseudomonas plays a central role in microbial networks, influencing keystone species despite being categorized as peripheral (interacting with 47 different taxa, 13 of which are keystone species). Anti‐microbiota vaccination targeting Pseudomonas and Lactobacillus yields distinct effects on tick fitness, with Pseudomonas vaccination significantly impacting female tick survival, while Lactobacillus significantly reduced oviposition and fertility. Microbiome changes post‐vaccination reveal diversity alterations, emphasizing the impact of vaccine choice. Community assembly dynamics and network robustness analyses highlight Pseudomonas' pivotal role, in influencing topological features and network resilience. The findings of the study provide comprehensive insights into the intricate dynamics of Om microbial networks and the potential of targeted microbiota‐driven vaccines for tick control. [ABSTRACT FROM AUTHOR]

African Swine Fever (ASF) is caused by a DNA virus (AFSV) maintained and transmitted by the Argasid ticks. The re-emergence of the disease in Africa coupled with its rapid spread globally is a threat to the pig industry, food security and livelihoods. The ecology and epidemiology of the ASFV sylvatic cycle, especially in the face of changing land use and land cover, further compounds the menace and impacts of this disease in Kenya. The study aimed to determine the occurrence and distribution of ASFV seroprevalence in warthog populations, the tick vectors and extent of tick infestation of warthog burrows, and the genotypes of ASFV in soft ticks in Kenya. Warthogs from different parts of Kenya were captured and venous blood was centrifuged to harvest sera. Warthog burrows were examined for their conditions and to extract ticks. Sera were analyzed for antibodies against ASFV using a commercial ELISA kit coated with p32 ASFV recombinant protein. Ticks were pooled, DNA extracted and the p72 gene of the ASFV was amplified by qPCR and conventional PCR. The overall seroprevalence of ASFV in warthogs was 87.5 %. A total of 228 warthog burrows were examined and 2154 argasid ticks were extracted from the burrows. Tick pools from Kigio Farm and Lewa Wildlife Conservancies were ASFV-positive by qPCR and conventional PCR. ASFV was further confirmed by the Twist Comprehensive Viral Research Panel (TCVRP), which also identified the argasid ticks as Ornithodoros porcinus. The ticks were infected with virus genotype IX, and their occurrence overlaps with regions of previous ASF outbreaks in domestic pigs. Further, Viruses that could be tick endosymbionts/commensals or due to bloodmeal were detected in ticks by TCVRP; Porcine type-C oncovirus; Pandoravirus neocaledonia; Choristoneura fumiferana granulovirus; Enterobacteria phage p7; Leporid herpesvirus 4 isolate; 5; Human Lymphotropic virus; Human herpesvirus 5. In conclusion, our results suggest that infected Ornithodoros spp. seems to have a rich virome, which has not been explored but could be exploited to inform ASF control in Kenya. Further, the ecology of Ornithodoros spp. and burrow-use dynamics are complex and more studies are needed to understand these dynamics, specifically in the spread of ASFV at the interface of wild and domestic pigs. Further, our results provide evidence of genotype IX ASFV sylvatic cycle which through O. porcinus tick transmission has resulted in high exposure of adult common warthogs. Finally, the co-circulation of ASFV genotype IX in the same location with past ASF outbreaks in domestic pigs and presently in ticks brings to focus the role of the interface and ticks on virus transmission to pigs and warthogs.

Ornithodoros erraticus and Ornithodoros moubata ticks are the main vectors of the agents of human relapsing fever (TBRF) and African swine fever (ASF) in the Mediterranean Basin and Africa, respectively. Tick ​​saliva is crucial for complete tick feeding and pathogen transmission, as it contains numerous molecules such as proteins, lipids, and non-coding RNAs (ncRNA) including microRNAs (miRNA). MiRNAs are small ncRNAs capable of regulating the expression of their target messenger RNA (mRNA) leading to degradation or inhibition of its translation into protein. Research on miRNAs from ixodid ticks has revealed that miRNAs are involved in the regulation of different physiological processes of ticks, as well as in the modulation of host gene expression, immune response to tick bite and pathogen transmission. Regarding argasid ticks, there is not information about their miRNAs or their potential involvement in tick physiology and/or in the regulation of the tick-host-pathogen interactions. The aim of this work was to profile the miRNAs expressed in the saliva of O. erraticus and O. moubata, and the in silico prediction and functional analysis of their target genes in the swine host. As a whole, up to 72 conserved miRNAs families were identified in both species: 35 of them were shared and 23 and 14 families were unique to O. erraticus and O. moubata, respectively. The most abundant miRNAs families were mir-1, mir-10 and let-7 in O. erraticus and let-7, mir-252, mir-10 in O. moubata. Four miRNAs sequences of each species were validated by RT-qPCR confirming their presence in the saliva. Target gene prediction in the host (Sus scrofa) and functional analysis showed that the selected miRNAs are mainly involved in processes related to signal transduction, regulation of mRNA transcription and gene expression, synapse regulation, immune response, angiogenesis and vascular development. These results suggest that miRNAs could play an important role at the tick-host interface, providing new insights into this complex relationship that may contribute to a more precise selection of tick molecules for the development of therapeutic and immune strategies to control tick infestations and tick-borne pathogens.

Introduction: Ornithodoros erraticus and Ornithodoros moubata are the main vectors of African swine fever virus (ASFV) and the human relapsing fever spirochetes Borrelia hispanica and Borrelia crocidurae in the Mediterranean region and Borrelia duttoni in continental Africa. Manipulation of the tick microbiome has been shown to reduce vector fitness and competence in tick vectors, suggesting that the identification of key microbial players associated with tick tissues can inform interventions such as anti-microbiota vaccines to block pathogen development in the midgut and/or salivary glands. Methods: In this study, we analyzed and compared the microbiome of the salivary glands and midgut of O. erraticus and O. moubata. For the taxonomic and functional characterization of the tissue-specific microbiome, we used 16S rRNA amplicon sequencing and prediction of metabolic profiles using PICRUSt2. Cooccurrence networks were built to characterize the community assembly and identify keystone taxa in each tick species. Results: Our results revealed differences in the composition, diversity, and assembly of the bacterial microbiome of salivary glands and midgut within each tick species, but differences were more noticeable in O. moubata. Differences were also found in the microbiome of each tissue, salivary gland and midgut, between species. However, the 'Core Association Networks (CAN)' analysis revealed conserved patterns of interacting taxa in tissues within and between tick species. Different keystone taxa were identified in O. erraticus and O. moubata tissues, but Muribaculaceae and Alistipes were found as keystone taxa in the salivary glands of both tick species which justifies their use as anti-microbiota vaccine candidates to alter the microbiome and reduce tick fitness and/or block pathogen transmission. The high similarity of predicted metabolic pathways profiles between tissues of the two tick species suggests that taxonomic variability of the microbiome is not associated with significant changes in microbial functional profiles. Conclusion: We conclude that the taxonomic structure of the microbiome in O. erraticus and O. moubata is tissue-specific, suggesting niche partitioning of bacterial communities associated to these soft ticks. However, shared keystone taxa and conserved patterns of interacting taxa between tissues and tick species suggest the presence of key microbial players that could be used as anti-microbiota vaccine candidates to affect tick physiology and/or pathogen colonization. [ABSTRACT FROM AUTHOR]

IntroductionOrnithodoros erraticus and Ornithodoros moubata are the main vectors of African swine fever virus (ASFV) and the human relapsing fever spirochetes Borrelia hispanica and Borrelia crocidurae in the Mediterranean region and Borrelia duttoni in continental Africa. Manipulation of the tick microbiome has been shown to reduce vector fitness and competence in tick vectors, suggesting that the identification of key microbial players associated with tick tissues can inform interventions such as anti-microbiota vaccines to block pathogen development in the midgut and/or salivary glands.MethodsIn this study, we analyzed and compared the microbiome of the salivary glands and midgut of O. erraticus and O. moubata. For the taxonomic and functional characterization of the tissue-specific microbiome, we used 16S rRNA amplicon sequencing and prediction of metabolic profiles using PICRUSt2. Co-occurrence networks were built to characterize the community assembly and identify keystone taxa in each tick species.ResultsOur results revealed differences in the composition, diversity, and assembly of the bacterial microbiome of salivary glands and midgut within each tick species, but differences were more noticeable in O. moubata. Differences were also found in the microbiome of each tissue, salivary gland and midgut, between species. However, the ‘Core Association Networks (CAN)’ analysis revealed conserved patterns of interacting taxa in tissues within and between tick species. Different keystone taxa were identified in O. erraticus and O. moubata tissues, but Muribaculaceae and Alistipes were found as keystone taxa in the salivary glands of both tick species which justifies their use as anti-microbiota vaccine candidates to alter the microbiome and reduce tick fitness and/or block pathogen transmission. The high similarity of predicted metabolic pathways profiles between tissues of the two tick species suggests that taxonomic variability of the microbiome is not associated with significant changes in microbial functional profiles.ConclusionWe conclude that the taxonomic structure of the microbiome in O. erraticus and O. moubata is tissue-specific, suggesting niche partitioning of bacterial communities associated to these soft ticks. However, shared keystone taxa and conserved patterns of interacting taxa between tissues and tick species suggest the presence of key microbial players that could be used as anti-microbiota vaccine candidates to affect tick physiology and/or pathogen colonization.

Introduction: We developed a new simple method to assess the composition of proteinaceous components in the saliva of Ornithodoros moubata, the main vehicle for pathogen transmission and a likely source of bioactive molecules acting at the tick-vertebrate host interface. To collect naturally expectorated saliva from the ticks we employed an artificial membrane feeding technique using a simple, chemically defined diet containing phagostimulants and submitted native saliva samples collected in this way for liquid chromatographymass spectrometry (LC-MS) analysis. These experiments were conducted with groups of uninfected ticks as well as with O. moubata infected with B. duttonii. The ticks exhibited a fair feeding response to the tested diet with engorgement rates reaching as high as 60-100% of ticks per feeding chamber. The LC-MS analysis identified a total of 17 and 15 proteins in saliva samples from the uninfected and infected O. moubata nymphs, respectively. Importantly, the analysis was sensitive enough to detect up to 9 different proteins in the samples of saliva containing diet upon which as few as 6 nymphal ticks fed during the experiments. Some of the proteins recognized in the analysis are well known for their immunomodulatory activity in a vertebrate host, whereas others are primarily thought of as structural or "housekeeping" proteins and their finding in the naturally expectorated tick saliva confirms that they can be secreted and might serve some functions at the tick-host interface. Most notably, some of the proteins that have long been suspected for their importance in the vector-pathogen interactions of Borrelia spirochetes were detected only in the samples from infected ticks, suggesting that their expression was altered by the persistent colonization of the tick's salivary glands by spirochetes. The simple method described herein is an important addition to the toolbox available to study the vector-host-pathogen interactions in the rapidly feeding soft ticks. [ABSTRACT FROM AUTHOR]

Relapsing fever (RF) is an arthropod-borne disease caused by Borrelia spirochete, which is one of the major public health concerns in endemic regions including Africa. However, information on Borrelia spirochetes is limited in Zambia. Here, we investigate the Borrelia spirochetes harbored by Ornithodoros ticks in Zambian National Parks. We analyzed 182 DNA samples pooled from 886 Ornithodoros ticks. Of these, 43 tested positive, and their sequence revealed that the ticks harbored both Old and New World RF borreliae. This research presents the first evidence of Old-World RF borreliae in Zambia. The New World RF borreliae detected herein differed from the Candidatus Borrelia fainii previously reported in Zambia and were closely related to the pathogenic Borrelia sp. VS4 identified in Tanzania. Additionally, Borrelia theileri was recently reported in Zambia. Hence, at least four different Borrelia species occur in Zambia, and the organisms causing relapsing fever there might be more complex than previously thought. We empirically confirmed that real-time PCR with TaqMan minor groove binder probes accurately and simultaneously detected both Old and New World RF. In this manner, they could facilitate quantitative analyses of both types of RF borreliae. Subsequent investigations should endeavor to isolate the aforementioned Borrelia spp. and perform serosurveys on patients with RF. [ABSTRACT FROM AUTHOR]

Introduction: We developed a new simple method to assess the composition of proteinaceous components in the saliva of Ornithodoros moubata, the main vehicle for pathogen transmission and a likely source of bioactive molecules acting at the tick-vertebrate host interface. To collect naturally expectorated saliva from the ticks we employed an artificial membrane feeding technique using a simple, chemically defined diet containing phagostimulants and submitted native saliva samples collected in this way for liquid chromatography-mass spectrometry (LC-MS) analysis. These experiments were conducted with groups of uninfected ticks as well as with O. moubata infected with B. duttonii. The ticks exhibited a fair feeding response to the tested diet with engorgement rates reaching as high as 60-100% of ticks per feeding chamber. The LC-MS analysis identified a total of 17 and 15 proteins in saliva samples from the uninfected and infected O. moubata nymphs, respectively. Importantly, the analysis was sensitive enough to detect up to 9 different proteins in the samples of saliva containing diet upon which as few as 6 nymphal ticks fed during the experiments. Some of the proteins recognized in the analysis are well known for their immunomodulatory activity in a vertebrate host, whereas others are primarily thought of as structural or “housekeeping” proteins and their finding in the naturally expectorated tick saliva confirms that they can be secreted and might serve some functions at the tick-host interface. Most notably, some of the proteins that have long been suspected for their importance in the vector-pathogen interactions of Borrelia spirochetes were detected only in the samples from infected ticks, suggesting that their expression was altered by the persistent colonization of the tick’s salivary glands by spirochetes. The simple method described herein is an important addition to the toolbox available to study the vector-host-pathogen interactions in the rapidly feeding soft ticks.

Initiation of vitellogenesis by blood feeding is essential for egg maturation in ticks. Nutrients derived from the blood meal are utilized by female ticks to synthesize the yolk protein precursor vitellogenin (Vg). Engorged Ornithodoros moubata ticks can synthesize Vg whether mated or virgin, thus O. moubata is an excellent model for studying the relative roles of blood feeding and mating in tick vitellogenesis. Injection of rapamycin into engorged O. moubata resulted in a reduction of ovarian growth and yolk accumulation in the oocytes of mated females. OmVg expression in the midgut and fat body and protein concentrations in the hemolymph significantly decreased in mated ticks after injection with rapamycin, indicating that inhibition of the nutrient‐sensing target of rapamycin (TOR) pathway disrupts egg maturation at the levels of Vg expression and synthesis. These results suggest that the TOR‐signaling pathway induces vitellogenesis in response to nutritional stimulation after a blood meal in O. moubata and is functionally independent of the mating‐induced pathway. [ABSTRACT FROM AUTHOR]

Abstract Background The argasid tick Ornithodoros moubata is the main vector in mainland Africa of African swine fever virus and the spirochete Borrelia duttoni, which causes human relapsing fever. The elimination of populations of O. moubata would contribute to the prevention and control of these two serious diseases. Anti-tick vaccines are an eco-friendly and sustainable means of eliminating tick populations. Tick saliva forms part of the tick-host interface, and knowledge of its composition is key to the identification and selection of vaccine candidate antigens. The aim of the present work is to increase the body of data on the composition of the saliva proteome of adult O. moubata ticks, particularly of females, since in-depth knowledge of the O. moubata sialome will allow the identification and selection of novel salivary antigens as targets for tick vaccines. Methods We analysed samples of female and male saliva using two different mass spectrometry (MS) approaches: data-dependent acquisition liquid chromatography-tandem MS (LC–MS/MS) and sequential window acquisition of all theoretical fragment ion spectra–MS (SWATH-MS). To maximise the number of proteins identified, a proteomics informed by transcriptomics analysis was applied using the O. moubata salivary transcriptomic dataset previously obtained by RNA-Seq. Results SWATH-MS proved to be superior to LC–MS/MS for the study of female saliva, since it identified 61.2% more proteins than the latter, the reproducibility of results was enhanced with its use, and it provided a quantitative picture of salivary components. In total, we identified 299 non-redundant proteins in the saliva of O. moubata, and quantified the expression of 165 of these in both male and female saliva, among which 13 were significantly overexpressed in females and 40 in males. These results indicate important quantitative differences in the saliva proteome between the sexes. Conclusions This work expands our knowledge of the O. moubata sialome, particularly that of females, by increasing the number of identified novel salivary proteins, which have different functions at the tick–host feeding interface. This new knowledge taken together with information on the O. moubata sialotranscriptome will allow a more rational selection of salivary candidates as antigen targets for tick vaccine development. Graphical Abstract

• Detection of African Swine Fever Virus (ASFV) in Ornithodoros porcinus in Kenya. • Ornithodoros porcinus is a vector of Genotype IX ASFV. • Common warthogs have a high exposure to ASFV. • Twist Comprehensive Viral Research Panel (TCVRP) can be used for detection of DNA viruses as well as barcoding of. tick species • Ornithodoros porcinus seems to have a rich virome, which is yet to be explored but could be exploited to inform ASF control in Africa. African Swine Fever (ASF) is caused by a DNA virus (AFSV) maintained and transmitted by the Argasid ticks. The re-emergence of the disease in Africa coupled with its rapid spread globally is a threat to the pig industry, food security and livelihoods. The ecology and epidemiology of the ASFV sylvatic cycle, especially in the face of changing land use and land cover, further compounds the menace and impacts of this disease in Kenya. The study aimed to determine the occurrence and distribution of ASFV seroprevalence in warthog populations, the tick vectors and extent of tick infestation of warthog burrows, and the genotypes of ASFV in soft ticks in Kenya. Warthogs from different parts of Kenya were captured and venous blood was centrifuged to harvest sera. Warthog burrows were examined for their conditions and to extract ticks. Sera were analyzed for antibodies against ASFV using a commercial ELISA kit coated with p32 ASFV recombinant protein. Ticks were pooled, DNA extracted and the p72 gene of the ASFV was amplified by qPCR and conventional PCR. The overall seroprevalence of ASFV in warthogs was 87.5 %. A total of 228 warthog burrows were examined and 2154 argasid ticks were extracted from the burrows. Tick pools from Kigio Farm and Lewa Wildlife Conservancies were ASFV-positive by qPCR and conventional PCR. ASFV was further confirmed by the Twist Comprehensive Viral Research Panel (TCVRP), which also identified the argasid ticks as Ornithodoros porcinus. The ticks were infected with virus genotype IX, and their occurrence overlaps with regions of previous ASF outbreaks in domestic pigs. Further, Viruses that could be tick endosymbionts/commensals or due to bloodmeal were detected in ticks by TCVRP; Porcine type-C oncovirus; Pandoravirus neocaledonia; Choristoneura fumiferana granulovirus ; Enterobacteria phage p7; Leporid herpesvirus 4 isolate; 5; Human Lymphotropic virus; Human herpesvirus 5. In conclusion, our results suggest that infected Ornithodoros spp. seems to have a rich virome, which has not been explored but could be exploited to inform ASF control in Kenya. Further, the ecology of Ornithodoros spp. and burrow-use dynamics are complex and more studies are needed to understand these dynamics, specifically in the spread of ASFV at the interface of wild and domestic pigs. Further, our results provide evidence of genotype IX ASFV sylvatic cycle which through O. porcinus tick transmission has resulted in high exposure of adult common warthogs. Finally, the co-circulation of ASFV genotype IX in the same location with past ASF outbreaks in domestic pigs and presently in ticks brings to focus the role of the interface and ticks on virus transmission to pigs and warthogs. [Display omitted] [ABSTRACT FROM AUTHOR]

Ornithodoros moubata transmits African swine fever and human relapsing fever in Africa. The elimination of O. moubata populations from anthropic environments is expected to improve the prevention and control of these diseases. Tick vaccines have emerged as a sustainable method for tick control, and tick aquaporins (AQPs) are promising targets for tick vaccines due to their vital functions, immunogenicity and ease of access by neutralising host antibodies. This study aimed at the systematic identification of the AQPs expressed by O. moubata (OmAQPs) and their characterisation as vaccine targets. Therefore, AQP coding sequences were recovered from available transcriptomic datasets, followed by PCR amplification, cloning, sequence verification and the analysis of the AQP protein structure and epitope exposure. Seven OmAQPs were identified and characterised: six were aquaglyceroporins, and one was a water-specific aquaporin. All of these were expressed in the salivary glands and midgut and only three in the coxal glands. Epitope exposure analysis identified three extracellular domains in each AQP, which concentrate overlapping B and T cell epitopes, making them interesting vaccine targets. Based on these domain sequences, a set of ten antigenic peptides was designed, which showed adequate properties to be produced and tested in pilot vaccine trials. [ABSTRACT FROM AUTHOR]

Studies on tick-pathogen-host interactions are helping to identify candidates for vaccines against ticks and tick-borne diseases and to discover potent bioactive tick molecules. The tick midgut is the main tissue involved in blood feeding and, moreover, the first organ to have contact with pathogens ingested through the blood meal. As little is known about the molecular biology of feeding and tick defence mechanisms against microorganisms, but important for understanding vector-pathogen interactions, we explored the early transcriptional changes in the midgut of Ornithodoros moubata after feeding and in response to challenge with the relapsing-fever spirochete Borrelia duttonii using the Ion S5XL platform. Besides transcripts with metabolic function and immune-related transcripts we discovered numerous putative and uncharacterized protein sequences. Overall, our analyses support previous studies and provides a valuable reference database for further functional proteomic analysis of midgut proteins of O. moubata. [ABSTRACT FROM AUTHOR]

Relapsing fever (RF) is an arthropod-borne disease caused by Borrelia spirochete, which is one of the major public health concerns in endemic regions including Africa. However, information on Borrelia spirochetes is limited in Zambia. Here, we investigate the Borrelia spirochetes harbored by Ornithodoros ticks in Zambian National Parks. We analyzed 182 DNA samples pooled from 886 Ornithodoros ticks. Of these, 43 tested positive, and their sequence revealed that the ticks harbored both Old and New World RF borreliae. This research presents the first evidence of Old-World RF borreliae in Zambia. The New World RF borreliae detected herein differed from the Candidatus Borrelia fainii previously reported in Zambia and were closely related to the pathogenic Borrelia sp. VS4 identified in Tanzania. Additionally, Borrelia theileri was recently reported in Zambia. Hence, at least four different Borrelia species occur in Zambia, and the organisms causing relapsing fever there might be more complex than previously thought. We empirically confirmed that real-time PCR with TaqMan minor groove binder probes accurately and simultaneously detected both Old and New World RF. In this manner, they could facilitate quantitative analyses of both types of RF borreliae. Subsequent investigations should endeavor to isolate the aforementioned Borrelia spp. and perform serosurveys on patients with RF.

Background: The argasid tick Ornithodoros moubata is the main vector in mainland Africa of African swine fever virus and the spirochete Borrelia duttoni, which causes human relapsing fever. The elimination of populations of O. moubata would contribute to the prevention and control of these two serious diseases. Anti-tick vaccines are an eco-friendly and sustainable means of eliminating tick populations. Tick saliva forms part of the tick-host interface, and knowledge of its composition is key to the identification and selection of vaccine candidate antigens. The aim of the present work is to increase the body of data on the composition of the saliva proteome of adult O. moubata ticks, particularly of females, since in-depth knowledge of the O. moubata sialome will allow the identification and selection of novel salivary antigens as targets for tick vaccines. Methods: We analysed samples of female and male saliva using two different mass spectrometry (MS) approaches: data-dependent acquisition liquid chromatography-tandem MS (LC–MS/MS) and sequential window acquisition of all theoretical fragment ion spectra–MS (SWATH-MS). To maximise the number of proteins identified, a proteomics informed by transcriptomics analysis was applied using the O. moubata salivary transcriptomic dataset previously obtained by RNA-Seq. Results: SWATH-MS proved to be superior to LC–MS/MS for the study of female saliva, since it identified 61.2% more proteins than the latter, the reproducibility of results was enhanced with its use, and it provided a quantitative picture of salivary components. In total, we identified 299 non-redundant proteins in the saliva of O. moubata, and quantified the expression of 165 of these in both male and female saliva, among which 13 were significantly overexpressed in females and 40 in males. These results indicate important quantitative differences in the saliva proteome between the sexes. Conclusions: This work expands our knowledge of the O. moubata sialome, particularly that of females, by increasing the number of identified novel salivary proteins, which have different functions at the tick–host feeding interface. This new knowledge taken together with information on the O. moubata sialotranscriptome will allow a more rational selection of salivary candidates as antigen targets for tick vaccine development. [ABSTRACT FROM AUTHOR]

Ornithodoros moubata transmits African swine fever and human relapsing fever in Africa. The elimination of O. moubata populations from anthropic environments is expected to improve the prevention and control of these diseases. Tick vaccines have emerged as a sustainable method for tick control, and tick aquaporins (AQPs) are promising targets for tick vaccines due to their vital functions, immunogenicity and ease of access by neutralising host antibodies. This study aimed at the systematic identification of the AQPs expressed by O. moubata (OmAQPs) and their characterisation as vaccine targets. Therefore, AQP coding sequences were recovered from available transcriptomic datasets, followed by PCR amplification, cloning, sequence verification and the analysis of the AQP protein structure and epitope exposure. Seven OmAQPs were identified and characterised: six were aquaglyceroporins, and one was a water-specific aquaporin. All of these were expressed in the salivary glands and midgut and only three in the coxal glands. Epitope exposure analysis identified three extracellular domains in each AQP, which concentrate overlapping B and T cell epitopes, making them interesting vaccine targets. Based on these domain sequences, a set of ten antigenic peptides was designed, which showed adequate properties to be produced and tested in pilot vaccine trials.

Studies on tick-pathogen-host interactions are helping to identify candidates for vaccines against ticks and tick-borne diseases and to discover potent bioactive tick molecules. The tick midgut is the main tissue involved in blood feeding and, moreover, the first organ to have contact with pathogens ingested through the blood meal. As little is known about the molecular biology of feeding and tick defence mechanisms against microorganisms, but important for understanding vector-pathogen interactions, we explored the early transcriptional changes in the midgut of Ornithodoros moubata after feeding and in response to challenge with the relapsing-fever spirochete Borrelia duttonii using the Ion S5XL platform. Besides transcripts with metabolic function and immune-related transcripts we discovered numerous putative and uncharacterized protein sequences. Overall, our analyses support previous studies and provides a valuable reference database for further functional proteomic analysis of midgut proteins of O. moubata.

Ticks (Chelicerata, Ixodida) are blood-feeding ectoparasites believed to have evolved at least about 120 millions of years ago and found worldwide. However, many aspects of their unique life cycle and anatomy, including their mechanical properties, remain to be understood. Here, we compared the mechanical properties of the cuticle of the argasid tick Ornithodoros moubata to those of two species of ixodid tick, Amblyomma hebraeum and Ixodes pacificus that we explored in our earlier studies of the tick exoskeleton. Significant differences were expected given the substantial difference in life cycle, including a five-fold increase during the repeated adult blood meal for female O. moubata vs. 70- to 120-fold during the single feeding of the adult female A. hebraeum and I. pacificus. We demonstrate here that the layered structure and mechanical properties (stiffness and viscosity) of the cuticle show minor differences, but the difference in cuticle thickness is substantial. Ductility is lost during feeding; reduced pH restores ductility. Previous work suggests that this occurs in vivo in engorged ixodid ticks; there is no evidence of this occurring in vivo in O. moubata. Thinning of cuticle in O. moubata fed females is consistent with the predicted stretch of cuticle due to the blood meal; there is no evidence of cuticle synthesis during the short feeding period. Dimensional analysis suggests that the soft feel of argasid ticks is related to cuticle thickness, not cuticle stiffness. Relative to argasid ticks, the hard ixodid ticks accommodate a ca. 20-fold higher size of blood meal by starting with a thicker cuticle and growing much additional cuticle during engorgement. [ABSTRACT FROM AUTHOR]

African swine fever (ASF) causes persistent outbreaks in endemic and non‐endemic regions in Zambia. However, the epidemiology of the disease is poorly understood, particularly during the inter‐epidemic periods. We conducted surveillance for ASF in asymptomatic domestic pigs and soft ticks in selected Zambian provinces. While serum samples (n = 1,134) were collected from crossbred pigs from all study sites between 2014 and 2017, whole blood (n = 300) was collected from both crossbred and indigenous pigs in Eastern Province (EP) in 2017. Soft ticks were collected from Mosi‐oa‐Tunya National Park in Southern Province (SP) in 2019. Sera were screened for antibodies against ASF by ELISA while genome detection in whole blood and soft ticks was conducted by PCR. Ticks were identified morphologically and by phylogenetic analysis of the 16S rRNA gene. Seroprevalence was highest in EP (50.9%, 95% CI [47.0–54.9]) compared to significantly lower rates in SP (2.9%, 95% CI [1.6–5.1]). No antibodies to ASFV were detected in Lusaka Province. In EP, the prevalence of ASFV genome was 11.7% (35/300), significantly higher (OR = 6.2, 95% CI [2.4–16.6]) in indigenous pigs compared to crossbred pigs. The pooled prevalence of ASFV genome in ticks was 11.0%, 95% CI [8.5–13.9]. Free‐range husbandry system was the only factor that was significantly associated with seropositive (p <.0001, OR = 39.3) and PCR‐positive results (p <.001, OR = 5.7). Phylogenetically, based on the p72 gene, ASFV from Ornithodoros moubata ticks detected in this study belonged to genotype I, but they separated into two distinct clusters. Besides confirming ASF endemicity in EP and the presence of ASFV‐infected ticks in SP, these results provide evidence for exposure of domestic pigs to ASFV in non‐endemic regions during the inter‐epidemic period. [ABSTRACT FROM AUTHOR]

Abstract Background The argasid tick Ornithodoros moubata is the main African vector of the human relapsing fever agent Borrelia duttoni and the African swine fever virus. Together with saliva, the tick midgut forms part of the host-tick-pathogen interface, and numerous midgut proteins play key functions in the blood digestion-related process and the infection and transmission of pathogens. This work explores the composition of the midgut proteome of unfed and fed O. moubata females with the aim to complete the biological information already obtained from the midgut transcriptome and provide a more robust and comprehensive perspective of this biological system. Methods Midgut tissues taken from females before feeding and 48 h after feeding were subjected to LC/MS-MS analysis. After functional characterization and classification of the proteins identified, the differences in the proteome between unfed and fed females were analysed and discussed. Additionally, a detailed analysis of particular groups of proteins that are involved in the processes of nutrient digestion and responses to the oxidative stress was carried out. Results 1491 non-redundant tick proteins were identified: 1132 of them in the midgut of unfed ticks, 1138 in the midgut of fed ticks, and up to 779 shared by both physiological conditions. Overall, the comparative analysis of the midgut proteomes of O. moubata females before and after feeding did not reveal great differences in the number or class of proteins expressed, enzymatic composition or functional classification. Conclusions The hemoglobinolytic system in ixodids and argasids is very similar in spite of the fact that they display very different feeding and reproductive strategies. Although the main source of nutrients in ticks are proteins, lipids and carbohydrates also constitute significant nutritional sources and play an important part in the process of blood digestion. The genes and proteins involved in intracellular transport mechanisms, defensive responses, detoxifying responses and stress responses seem to be closely regulated, highlighting the complexity and importance of these processes in tick biology, which in turn assigns them a great interest as targets for therapeutic and immunological interventions.

• First profiles of salivary miRNAs expressed by argasid ticks are reported. • Up to 72 conserved miRNAs families were identified in two Ornithodoros species. • Target gene prediction show that miRNAs regulate tick-host relationships. • Functional analysis of miRNA-targeted genes may instruct tick vaccine development. Ornithodoros erraticus and Ornithodoros moubata ticks are the main vectors of the agents of human relapsing fever (TBRF) and African swine fever (ASF) in the Mediterranean Basin and Africa, respectively. Tick ​​saliva is crucial for complete tick feeding and pathogen transmission, as it contains numerous molecules such as proteins, lipids, and non-coding RNAs (ncRNA) including microRNAs (miRNA). MiRNAs are small ncRNAs capable of regulating the expression of their target messenger RNA (mRNA) leading to degradation or inhibition of its translation into protein. Research on miRNAs from ixodid ticks has revealed that miRNAs are involved in the regulation of different physiological processes of ticks, as well as in the modulation of host gene expression, immune response to tick bite and pathogen transmission. Regarding argasid ticks, there is not information about their miRNAs or their potential involvement in tick physiology and/or in the regulation of the tick-host-pathogen interactions. The aim of this work was to profile the miRNAs expressed in the saliva of O. erraticus and O. moubata , and the in silico prediction and functional analysis of their target genes in the swine host. As a whole, up to 72 conserved miRNAs families were identified in both species: 35 of them were shared and 23 and 14 families were unique to O. erraticus and O. moubata , respectively. The most abundant miRNAs families were mir-1, mir-10 and let-7 in O. erraticus and let-7, mir-252, mir-10 in O. moubata. Four miRNAs sequences of each species were validated by RT-qPCR confirming their presence in the saliva. Target gene prediction in the host (Sus scrofa) and functional analysis showed that the selected miRNAs are mainly involved in processes related to signal transduction, regulation of mRNA transcription and gene expression, synapse regulation, immune response, angiogenesis and vascular development. These results suggest that miRNAs could play an important role at the tick-host interface, providing new insights into this complex relationship that may contribute to a more precise selection of tick molecules for the development of therapeutic and immune strategies to control tick infestations and tick-borne pathogens. [Display omitted] [ABSTRACT FROM AUTHOR]

Epigenetic mechanisms have not been characterized in ticks despite their importance as vectors of human and animal diseases worldwide. Our investigation identifies and functionally characterizes the orthologue of S-adenosylmethionine (SAM) binding methyltransferase enzyme, disruptor of telomeric silencing 1-like (DOT1L) in Ornithodoros moubata (OmDOT1L), a soft tick vector for the relapsing fever pathogen Borrelia duttonii and the African swine fever virus. The OmDOT1L tertiary structure was predicted and compared to the Homo sapiens DOT1L which had been co-crystalized with SGC0946, a DOT1L-specific inhibitor. The amino acid residues crucial for SAM and SGC0946 binding conserved in most DOT1L sequences available, are also conserved in OmDOT1L. Quantitative PCR of Omdot1l during O. moubata life stages showed that transcripts were significantly upregulated in first-stage nymphs. O. moubata larvae exposed to SGC0946 displayed high mortality during molting to first-stage nymphs. Furthermore, a significant decrease in weight was observed in second-stage nymphs fed on recombinant OmDOT1L-immunized rabbits. In contrast, artificial blood feeding supplemented with SGC0946 did not affect survival and reproductive performance of adult female ticks. We concluded that OmDOT1L plays an essential role in the regulation of larval molting and the feeding of O. moubata second-stage nymphs.

Ornithodoros soft ticks are the only known vector and reservoir of the African swine fever virus, a major lethal infectious disease of Suidae. The co-feeding event for virus transmission and maintenance among soft tick populations has been poorly documented. We infected Ornithodoros moubata, a known tick vector in Africa, with an African swine fever virus strain originated in Africa, to test its ability to infect O. moubata through co-feeding on domestic pigs. In our experimental conditions, tick-to-tick virus transmission through co-feeding failed, although pigs became infected through the infectious tick bite.

Abstract Background Fluralaner is a novel isoxazoline eliciting both acaricidal and insecticidal activity through potent blockage of GABA- and glutamate-gated chloride channels. The aim of the study was to investigate the susceptibility of juvenile stages of common tick species exposed to fluralaner through either contact (Rhipicephalus sanguineus) or contact and feeding routes (Ornithodoros moubata). Methods Fluralaner acaricidal activity through both contact and feeding exposure was measured in vitro using two separate testing protocols. Acaricidal contact activity against Rhipicephalus sanguineus life stages was assessed using three minute immersion in fluralaner concentrations between 50 and 0.05 μg/mL (larvae) or between 1000 and 0.2 μg/mL (nymphs and adults). Contact and feeding activity against Ornithodoros moubata nymphs was assessed using fluralaner concentrations between 1000 to 10−4 μg/mL (contact test) and 0.1 to 10−10 μg/mL (feeding test). Activity was assessed 48 hours after exposure and all tests included vehicle and untreated negative control groups. Results Fluralaner lethal concentrations (LC50, LC90/95) were defined as concentrations with either 50%, 90% or 95% killing effect in the tested sample population. After contact exposure of R. sanguineus life stages lethal concentrations were (μg/mL): larvae - LC50 0.7, LC90 2.4; nymphs - LC50 1.4, LC90 2.6; and adults - LC50 278, LC90 1973. After exposure of O. moubata nymphs to fluralaner lethal concentrations were (μg/mL): contact exposure - LC50 720, LC95 1133; and feeding exposure- LC50 0.00007, LC95 0.09. Conclusions Fluralaner demonstrates potent in vitro acaricidal activity against all life stages of the brown dog tick, R.sanguineus. The testing of fluralaner contact and feeding routes using O. moubata nymphs demonstrates a high acaricidal activity in both exposure routes.

The African argasid tick Ornithodoros moubata transmits two important pathogens, the African swine fever virus and the spirochete Borrelia duttoni , the cause of human relapsing fever. To date, only conventional control measures such as widespread application of acaricides, strict control measures, and animal movement restrictions have been implemented to confine these diseases. Vaccines against tick infestations have the potential to be among the most efficacious interventions for the management of these diseases. Plasma membrane-associated proteins upregulated in tick midgut cells in response to blood feeding and digestion are thought to play vital functions in tick physiology and in the transmission of tick-borne pathogens. In addition, their antigenic extracellular regions are easily accessible to antibodies synthesised by immunised hosts, which makes them interesting targets for tick vaccine design. The mialomes (midgut transcriptomes and proteomes) of unfed O. moubata females and of engorged females at 48 h post-feeding have recently been obtained, providing a wealth of predicted midgut protein sequences. In the current study, these mialomes were screened using in silico tools to select predicted antigenic transmembrane proteins that were upregulated after feeding (516 proteins). The functionally annotatable proteins from this list (396 proteins) were then manually inspected following additional criteria in order to select a finite and easy-manageable number of candidate antigens for tick vaccine design. The extracellular antigenic regions of five of these candidates were obtained either as truncated recombinant proteins or as KLH-conjugated synthetic peptides, formulated in Freund’s adjuvant, and individually administered to rabbits to assess their immunogenicity and protective potential against infestations by O. moubata and the Iberian species Ornithodoros erraticus . All candidates were highly immunogenic, but provided low protection against the O. moubata infestations (ranging from 7% to 39%). Interestingly, all candidates except one also protected against infestations by O. erraticus , achieving higher efficacies against this species (from 20% to 66%). According to their protective potential, three of the five antigens tested (Om17, Om86 and OM99) were considered little suitable for use in tick vaccines, while the other two (OM85 and OM03) were considered useful antigens for tick vaccine development, deserving further studies. [ABSTRACT FROM AUTHOR]

Ornithodoros erraticus and Ornithodoros moubata are argasid tick vectors that transmit severe diseases such as African swine fever and human relapsing fever. Elimination of the synanthropic populations of these vectors would facilitate the control of these diseases. Fluralaner is a novel isoxazoline that selectively blocks the GABA- and glutamate-gated channels, providing potent insecticidal and acaricidal activity. The aim of the current study was to provide quantitative data on the susceptibility of males, females and third nymphal instar of O. erraticus and O. moubata to fluralaner through in vitro feeding exposure. Fluralaner activity against these developmental stages and species was assessed by feeding the ticks on ovine blood medicated with decreasing fluralaner concentrations between 1 and 10 −8 μg/mL. Tick mortality was measured at 4, 24 and 48 h and 1, 2 and 3 weeks post-feeding. Tests included solvent-treated and untreated blood controls. Fluralaner was extremely active against O. erraticus, with mean lethal concentrations 50 (LC 50 ) and 95 (LC 95 ) of 2.0 × 10 −8 and 5.4 × 10 −8 μg/mL, respectively. Fluralaner was also highly active against O. moubata , showing a mean LC 50 of 1.5 × 10 −6 μg/mL and a mean LC 95 of 1.8 × 10 −3 μg/mL. In the latter species, the most susceptible life stages were the females (LC 95 1.4 × 10 −4 μg/mL). Fluralaner demonstrated potent acaricidal activity against all developmental stages of O. erraticus and O. moubata tested, in the first 48 h after in vitro feeding. Therefore, fluralaner has the potential to provide very high acaricidal efficacy to multiple argasid tick species via feeding exposure and could be included as an acaricidal agent in integrated programmes for the control of argasid tick vectors and argasid tick-borne diseases. [ABSTRACT FROM AUTHOR]

Background: The argasid tick Ornithodoros moubata is the main African vector of the human relapsing fever agent Borrelia duttoni and the African swine fever virus. Together with saliva, the tick midgut forms part of the host-tickpathogen interface, and numerous midgut proteins play key functions in the blood digestion-related process and the infection and transmission of pathogens. This work explores the composition of the midgut proteome of unfed and fed O. moubata females with the aim to complete the biological information already obtained from the midgut transcriptome and provide a more robust and comprehensive perspective of this biological system. Methods: Midgut tissues taken from females before feeding and 48 h after feeding were subjected to LC/MS-MS analysis. After functional characterization and classification of the proteins identified, the differences in the proteome between unfed and fed females were analysed and discussed. Additionally, a detailed analysis of particular groups of proteins that are involved in the processes of nutrient digestion and responses to the oxidative stress was carried out. Results: 1491 non-redundant tick proteins were identified: 1132 of them in the midgut of unfed ticks, 1138 in the midgut of fed ticks, and up to 779 shared by both physiological conditions. Overall, the comparative analysis of the midgut proteomes of O. moubata females before and after feeding did not reveal great differences in the number or class of proteins expressed, enzymatic composition or functional classification. Conclusions: The hemoglobinolytic system in ixodids and argasids is very similar in spite of the fact that they display very different feeding and reproductive strategies. Although the main source of nutrients in ticks are proteins, lipids and carbohydrates also constitute significant nutritional sources and play an important part in the process of blood digestion. The genes and proteins involved in intracellular transport mechanisms, defensive responses, detoxifying responses and stress responses seem to be closely regulated, highlighting the complexity and importance of these processes in tick biology, which in turn assigns them a great interest as targets for therapeutic and immunological interventions. [ABSTRACT FROM AUTHOR]

The argasid tick Ornithodoros moubata is the main vector of the African swine fever and the human relapsing fever in Africa. As part of the host-parasite-pathogen interface, the tick midgut expresses key proteins for tick survival and tick-borne pathogen transmission. Accordingly, midgut proteins are potential targets for the development of new drugs and vaccines aimed at tick control, and obtaining proteomic and transcriptomic data from the O. moubata midgut would facilitate the identification of such target candidates. With this aim, we have assembled and characterized the midgut transcriptome of O. moubata females before and 48 h after a blood meal, and identified the genes that are differentially expressed in the midgut after feeding. Overall, 23,863 transcripts were obtained, and of them, 9,164 were identified and annotated. The most represented molecular functions were catalytic and binding activities, and the most represented biological processes were metabolic, cellular and single-organism processes. KEGG analysis of the annotated sequences assigned up to 3,053 of them to 130 active pathways, among which, the top 30 pathways were mostly metabolic routes. Differential expression analysis between unfed and fed ticks detected 8,026 Differentially Expressed Genes (DEGs), 4,093 up-regulated and 3,933 down-regulated, respectively. The biological significance of these DEGs was further investigated using the KEEG, Pfam and GO databases. The functional groups of the genes/proteins predicted to be involved in the processes of blood digestion, nutrient transport and metabolism, and in responses related to defence and oxidative stress are discussed in more detail. This work reports the first midgut transcriptome analysis of an argasid tick species, and provides a wealth of novel molecular information about the argasid machinery involved in blood feeding and digestion. This information represents a starting point for the development of alternative strategies for tick control. [ABSTRACT FROM AUTHOR]

African swine fever (ASF) has been reported in South Africa since the early 20th century. The disease has been controlled and confined to northern South Africa over the past 80 years by means of a well-defined boundary line, with strict control measures and movement restrictions north of this line. In 2012, the first outbreak of ASF outside the ASF control zone since 1996 occurred. The objective of this study was to evaluate the current relevance of the ASF control line as a demarcation line between endemic ASF (north) areas and ASF-free (south) area and to determine whether there was a need to realign its trajectory, given the recent outbreaks of ASF, global climate changes and urban development since the line’s inception. A study of ASF determinants was conducted in an area 20 km north and 20 km south of the ASF control line, in Limpopo, Mpumalanga, North West and Gauteng provinces between May 2008 and September 2012. The study confirmed that warthogs, warthog burrows and the soft tick reservoir, Ornithodoros moubata, are present south of the ASF control line, but no virus or viral DNA was detected in these ticks. There appears to be an increasing trend in the diurnal maximum temperature and a decrease in humidity along the line, but the impact of these changes is uncertain. No discernible changes in minimum temperatures and average rainfall along the disease control line were observed between 1992 and 2014. Even though the reservoirs were found south of the ASF boundary line, the study concluded that there was no need to realign the trajectory of the ASF disease control line, with the exception of Limpopo Province. However, the provincial surveillance programmes for the reservoir, vector and ASF virus south of this line needs to be maintained and intensified as changing farming practices may favour the spread of ASF virus beyond the control line. Keywords: African swine fever; warthog burrow; Ornithodoros moubata;control line

Background: African swine fever (ASF) causes severe socio-economic impacts due to high mortality and trade restrictions. Many risk factors of ASF have been identified at farm level. However, understanding the risk factors, especially wild suid hosts, determining ASF transmission at regional level remains limited. Methods: Based on ASF outbreak data in domestic pigs during 2006-2014, we here tested, separately for West and East Africa, which risk factors were linked to ASF presence at a regional level, using generalized linear mixed models. Results: Our results show that ASF infections in the preceding year was an important predictor for ASF presence in both West and East Africa. Both pig density and human density were positively associated with ASF presence in West Africa. In East Africa, ASF outbreaks in domestic pigs were also correlated with higher percentages of areas occupied by giant forest hogs and by high-tick-risk areas. Conclusions: Our results suggest that regional ASF risk in East Africa and in West Africa were associated with different sets of risk factors. Regional ASF risk in West Africa mainly followed the domestic cycle, whereas the sylvatic cycle may influence regional ASF risk in East Africa. With these findings, we contribute to the better understanding of the risk factors of ASF occurrence at regional scales that may aid the implementation of effective control measures. [ABSTRACT FROM AUTHOR]

Recently obtained evidence indicated that an orthologue of the O. savignyi TSGP4 salivary lipocalin was present in the saliva of O. moubata . TSGP4 is known to act as a cysteinyl leukotrienes scavenger helping in the prevention of inflammation and oedema at the tick bite site. Since this function seems to be crucial for successful tick feeding, the novel O. moubata TSGP4 turned into a potential vaccine target. The purposes of the current work were: (i) to clone and characterize the O. moubata TSGP4 and, (ii) to produce it as recombinant to evaluate its protective efficacy as vaccine antigen. The results of these experiments indicated that the O. moubata TSGP4 shows high sequence and structural identity with the O. savignyi orthologue suggesting identical function in the physiology of the tick-host relationship. The mature native TSGP4 is not immunogenic when it is inoculated to host with tick saliva during feeding, but host vaccination with the recombinant protein TSGP4 in Freund’s adjuvants induced strong humoral immune responses that recognized both the recombinant and native TSGP4 and protected the host with a 14.1% efficacy. So, the O. moubata TSGP4 can be considered a silent salivary antigen; however, in the light of the current results, its inclusion in the current repertory of protective antigens to be targeted by anti-tick vaccines could be controversial. [ABSTRACT FROM AUTHOR]

Ornithodoros moubata is the main vector of the pathogens causing African swine fever and human relapsing fever in Africa. The development of an efficient vaccine against this tick would facilitate its control and the prevention of the diseases it transmits to a considerable extent. Previous efforts to identify vaccine target candidates led us to the discovery of novel salivary proteins that probably act as anti-haemostatics at the host-tick interface, including a secreted phospholipase A 2 (PLA2), a 7DB-like protein (7DB-like), a riboprotein 60S L10 (RP-60S), an apyrase (APY), and a new platelet aggregation inhibitor peptide, designated mougrin (MOU). In this work, the corresponding recombinant proteins were expressed in Escherichia coli and their individual vaccine efficacy was tested in rabbit vaccination trials. All of them, except the less immunogenic RP-60S, induced strong humoral responses that reduced tick feeding and survival, providing vaccine efficacies of 44.2%, 43.2% and 27.2%, 19.9% and 17.3% for PLA2, APY, MOU, RP-60S and 7DB-like, respectively. In the case of the more protective recombinant antigens (PLA2, APY and MOU), the immunodominant protective linear B-cell epitopes were identified and their combined vaccine efficacy was tested in a second vaccine trial using different adjuvants. In comparison with the best efficacy of individual antigens, the multicomponent vaccine increased vaccine efficacy by 13.6%, indicating additive protective effects rather than a synergistic effect. Tick saliva inoculated during natural tick-host contacts had a boosting effect on vaccinated animals, increasing specific antibody levels and protection. [ABSTRACT FROM AUTHOR]

Ecdysteroidogenesis is essential for arthropod development and reproduction. Although the importance of ecdysteroids has been demonstrated, there is little information on the sites and enzymes for synthesis of ecdysteroids from Chelicerates. Ecdysteroid functions have been well studied in the soft tick Ornithodoros moubata, making this species an excellent candidate for elucidating ecdysteroidogenesis in Chelicerates. Results showed that O. moubata has at least two ecdysteroidogenic enzymes, Spook (OmSpo) and Shade (OmShd). RNAi showed both enzymes were required for ecdysteroidogenesis. Enzymatic assays demonstrated OmShd has the conserved functions of ecdysone 20-hydroxylase. OmSpo showed specific expression in the ovaries of final nymphal and adult stages, indicating O. moubata utilizes the ovary as an ecdysteroidogenic tissue instead of specific tissues as seen in other arthropods. On the other hand, OmShd expression was observed in various tissues including the midgut, indicating functional ecdysteroids can be produced in these tissues. In nymphal stages, expression of both OmSpo and OmShd peaked before molting corresponding with high ecdysteroid titers in the hemolymph. In fed adult females, OmSpo expression peaked at 8–10 days after engorgement, while OmShd expression peaked immediately after engorgement. Mated females showed more frequent surges of OmShd than virgin females. These results indicate that the regulation of synthesis of ecdysteroids differs in nymphs and adult females, and mating modifies adult female ecdysteroidogenesis. This is the first report to focus on synthesis of ecdysteroids in ticks and provides essential knowledge for understanding the evolution of ecdysteroidogenesis in arthropods. [ABSTRACT FROM AUTHOR]