Production of the yolk protein precursor vitellogenin is mediated by target of rapamycin (TOR) in the soft tick Ornithodoros moubata (Acari: Argasidae).

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.

Sialotranscriptomics of the argasid tick Ornithodoros moubata along the trophogonic cycle.

The argasid tick Ornithodoros moubata is the main vector of human relapsing fever (HRF) and African swine fever (ASF) in Africa. Salivary proteins are part of the host-tick interface and play vital roles in the tick feeding process and the host infection by tick-borne pathogens; they represent interesting targets for immune interventions aimed at tick control. The present work describes the transcriptome profile of salivary glands of O. moubata and assesses the gene expression dynamics along the trophogonic cycle using Illumina sequencing. De novo transcriptome assembling resulted in 71,194 transcript clusters and 41,011 annotated transcripts, which represent 57.6% of the annotation success. Most salivary gene expression takes place during the first 7 days after feeding (6,287 upregulated transcripts), while a minority of genes (203 upregulated transcripts) are differentially expressed between 7 and 14 days after feeding. The functional protein groups more abundantly overrepresented after blood feeding were lipocalins, proteases (especially metalloproteases), protease inhibitors including the Kunitz/BPTI-family, proteins with phospholipase A2 activity, acid tail proteins, basic tail proteins, vitellogenins, the 7DB family and proteins involved in tick immunity and defence. The complexity and functional redundancy observed in the sialotranscriptome of O. moubata are comparable to those of the sialomes of other argasid and ixodid ticks. This transcriptome provides a valuable reference database for ongoing proteomics studies of the salivary glands and saliva of O. moubata aimed at confirming and expanding previous data on the O. moubata sialoproteome.

A soft tick Ornithodoros moubata salivary protein OmCI is a potent inhibitor to prevent avian complement activation.

Complement is a key first line innate host defense system in the blood of vertebrates. Upon activation, this powerful defense mechanism can elicit inflammatory responses, lyse non-self-cells, or mark them for opsonophagocytic removal. Blood-feeding arthropods thus require the ability to block host complement activation in the bloodmeal to prevent undesired cell or tissue damage during feeding. The soft tick Ornithodoros moubata produces a complement inhibitory protein, OmCI. This protein binds to a mammalian complement protein C5 and blocks further activation of complement cascades, which results in the prevention of complement-mediated bacterial killing through membrane attack complex. Interestingly, the amino acids involved in OmCI binding are highly conserved among mammalian and avian C5, but the ability of this protein to inhibit the complement from birds remains unclear. Here we demonstrated that OmCI is capable of preventing quail complement-mediated erythrocyte lysis, inhibiting the capability of this animal's complement to eliminate a serum-sensitive Lyme disease bacterial strain. We also found that the ability of OmCI to inhibit quail complement-mediated killing of Lyme disease bacteria can be extended to different domestic and wild birds. Our results illustrate the utility of OmCI to block bird complement. These results provide the foundation for further use of this protein as a tool to study the molecular basis of avian complement and pathogen evasion to such a defense mechanism.

RNA-seq analysis of the salivary glands and midgut of the Argasid tick Ornithodoros rostratus.

Ornithodoros rostratus is a South American argasid tick which importance relies on its itchy bite and potential as disease vector. They feed on a wide variety of hosts and secrete different molecules in their saliva and intestinal content that counteract host defences and help to accommodate and metabolize the relatively large quantity of blood upon feeding. The present work describes the transcriptome profile of salivary gland (SG) and midgut (MG) of O. rostratus using Illumina sequencing. A total of 8,031 contigs were assembled and assigned to different functional classes. Secreted proteins were the most abundant in the SG and accounted for ~67% of all expressed transcripts with contigs with identity to lipocalins and acid tail proteins being the most representative. On the other hand, immunity genes were upregulated in MG with a predominance of defensins and lysozymes. Only 10 transcripts in SG and 8 in MG represented ~30% of all RNA expressed in each tissue and one single contig (the acid tail protein ORN-9707) represented ~7% of all expressed contigs in SG. Results highlight the functional difference of each organ and identified the most expressed classes and contigs of O. rostratus SG and MG.

Identification and characterization of a histamine-binding lipocalin-like molecule from the relapsing fever tick Ornithodoros turicata.

Lipocalins are low molecular weight membrane transporters that are abundantly expressed in the salivary glands and other tissues of ticks. In this study, we identified a lipocalin-like molecule, designated as otlip, from the soft ticks Ornithodoros turicata, the vector for the relapsing fever causing spirochete Borrelia turicatae. We noted that the expression of otlip was developmentally regulated, with adult ticks expressing significantly higher levels in comparison to the larvae or nymphal ticks. Expression of otlip was evident in both fed and unfed O. turicata ticks, with significantly increased expression in the salivary glands in comparison to the midgut or ovary tissues. High conservation of the biogenic amine-binding motif was evident in the deduced primary amino acid sequence of Otlip. Protein modelling of Otlip revealed conservation of most of the residues involved in binding histamine or serotonin ligand. In vitro assays demonstrated binding of recombinant Otlip with histamine. Furthermore, prediction of post-translational modifications revealed that Otlip contained phosphorylation and myristoylation sites. Taken together, our study not only provides evidence for the presence of a lipocalin-like molecule in O. turicata ticks but also suggests a role for this molecule in the salivary glands of this medically important vector.

A proteomic insight into the midgut proteome of Ornithodoros moubata females reveals novel information on blood digestion in argasid ticks.

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.

Gut transcriptome analysis on females of Ornithodoros mimon (Acari: Argasidae) and phylogenetic inference of ticks.

Ornithodoros mimon is an argasid tick that parasitizes bats, birds and opossums and is also harmful to humans. Knowledge of the transcripts present in the tick gut helps in understanding the role of vital molecules in the digestion process and parasite-host relationship, while also providing information about the evolution of arthropod hematophagy. Thus, the present study aimed to know and ascertain the main molecules expressed in the gut of argasid after their blood meal, through analysis on the gut transcriptome of engorged females of O. mimon using 454-based RNA sequencing. The gut transcriptome analysis reveals several transcripts associated with hemoglobin digestion, such as serine, cysteine, aspartic proteases and metalloenzymes. The phylogenetic analysis on the peptidases confirmed that most of them are clustered with other tick genes. We recorded the presence a cathepsin O peptidase-coding transcript in ticks. The topology of the phylogenetic inferences, based on transcripts of inferred families of homologues, was similar to that of previous reports based on mitochondrial genome and nuclear rRNA sequences. We deposited 2,213 sequence of O. mimon to the public databases. Our findings may help towards better understanding of important argasid metabolic processes, such as digestion, nutrition and immunity.

Gut transcriptome analysis on females of Ornithodoros mimon (Acari: Argasidae) and phylogenetic inference of ticks / Transcriptoma do intestino de fêmeas de Ornithodoros mimon (Acari: Argasidae) e inferência filogenética de carrapatos

Abstract Ornithodoros mimon is an argasid tick that parasitizes bats, birds and opossums and is also harmful to humans. Knowledge of the transcripts present in the tick gut helps in understanding the role of vital molecules in the digestion process and parasite-host relationship, while also providing information about the evolution of arthropod hematophagy. Thus, the present study aimed to know and ascertain the main molecules expressed in the gut of argasid after their blood meal, through analysis on the gut transcriptome of engorged females of O. mimon using 454-based RNA sequencing. The gut transcriptome analysis reveals several transcripts associated with hemoglobin digestion, such as serine, cysteine, aspartic proteases and metalloenzymes. The phylogenetic analysis on the peptidases confirmed that most of them are clustered with other tick genes. We recorded the presence a cathepsin O peptidase-coding transcript in ticks. The topology of the phylogenetic inferences, based on transcripts of inferred families of homologues, was similar to that of previous reports based on mitochondrial genome and nuclear rRNA sequences. We deposited 2,213 sequence of O. mimon to the public databases. Our findings may help towards better understanding of important argasid metabolic processes, such as digestion, nutrition and immunity.

Resumo Ornithodoros mimon é um carrapato argasídeo parasita de morcegos, aves e marsupiais, além de ser bastante agressivo aos humanos. O conhecimento dos transcritos presentes no intestino dos carrapatos auxilia no entendimento do papel de moléculas vitais no processo de digestão e na relação parasito-hospedeiro, além de fornecer também informações sobre a evolução dos artrópodes hematófagos. Desta maneira, o presente estudo teve como objetivo conhecer e identificar as principais moléculas expressas no intestino de uma espécie de carrapato argasídeo após o repasto sanguíneo, através de uma análise transcritômica descritiva do intestino de fêmeas ingurgitadas de O. mimon, utilizando um sequenciamento de RNA de nova geração da plataforma 454. Além de inferir a relação filogenética de carrapatos através de um conjunto de dados transcritômicos. O transcriptoma do intestino revelou diversos transcritos associados com a digestão da hemoglobina, como proteinases das classes serino, cisteína, aspártica e metalo. Registramos a presença de um transcrito de uma cisteína peptidase do tipo catepsina O em carrapatos. A inferência filogenética baseada em conjunto de dados transcritos homólogos tem uma resolução topológica similar a de outros conjuntos de dados moleculares. Foram depositados no banco de dados gênico público 2213 transcritos de O. mimon. Os achados obtidos no presente estudo podem contribuir para compreensão dos importantes processos, como digestão, nutrição e imunidade dos carrapatos da família Argasidae, além de fornecer informações sobre a filogenia da ordem Ixodida.

TSGP4 from Ornithodoros moubata: molecular cloning, phylogenetic analysis and vaccine efficacy of a new member of the lipocalin clade of cysteinyl leukotriene scavengers.

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.

Comparison of synganglion neuropeptides, neuropeptide receptors and neurotransmitter receptors and their gene expression in response to feeding in Ixodes scapularis (Ixodidae) vs. Ornithodoros turicata (Argasidae).

Illumina GAII high-throughput sequencing was used to compare expressed genes for female synganglion neuropeptides, neuropeptide receptors and neurotransmitter receptors of the soft tick Ornithodoros turicata with the hard tick Ixodes scapularis. Gene ontology molecular level three mapping revealed no significant differences amongst the same categories represented in O. turicata and I. scapularis. Transcripts predicting 22 neuropeptides or their receptors in the O. turicata synganglion were similar to annotations for 23 neuropeptides or receptors previously identified from I scapularis, with minor exceptions. A transcript predicting ecdysis triggering hormone receptor was identified in O. turicata; transcripts encoding for proprotein convertase and glycoprotein B were identified in both species. Transcripts predicting the same neurotransmitter receptors were found in the synganglion of both species. Gene expression of the transcripts showed numerous differences in response to feeding. Major differences were observed in expression of genes believed important in regulating slow vs. rapid feeding, blood water elimination, cuticle synthesis plasticity and in signalling reproductive activity. Although the glutamate receptor was strongly upregulated in both species, the gamma aminobutyric acid receptor, which inhibits glutamate, was upregulated significantly only in I. scapularis. These differences are consistent with the slow vs. rapid action of the pharyngeal pump in the two species.

Midgut proteome of an argasid tick, Ornithodoros erraticus: a comparison between unfed and engorged females.

BACKGROUND: The argasid tick Ornithodoros erraticus is the vector of African swine fever virus and of several Borrelia species that cause human relapsing fever in the Iberian Peninsula. The tick midgut is part of the ectoparasite-host interface and expresses proteins that are vital for the survival of the tick. Midgut proteins are therefore potential targets for drug and/or vaccine design aimed at the development of new strategies for tick control. Thus, the aim of this work was the characterization of the proteome of the O. erraticus midgut before and after a blood meal trying to elucidate the induced changes upon blood feeding. METHODS: Midgut tissues from unfed and engorged O. erraticus females were dissected and proteins were fractionated by centrifugation and SDS-PAGE, and the corresponding gel pieces analysed by LC-MS/MS. The identified proteins were classified according to their Protein Class and Molecular Function and the differences between fed and unfed specimens were analysed. RESULTS: Overall 555 tick proteins were identified: 414 in the midgut of the unfed specimens and 376 in the fed specimens, of which 235 were present in both groups. The proteins with catalytic, binding and structural functions were the most numerous and abundant, consistent with their role in the intracellular processing of the blood meal. The analysis of some groups of proteins putatively involved directly in blood meal digestion, including protein digestion (peptidase activity), iron metabolism, enzymes involved in oxidative stress and detoxification and membrane traffic and transport proteins, detected some differences between the fed and unfed ticks CONCLUSIONS: This work reports for the first time the collection and analysis of the midgut proteome of an argasid tick species and provides molecular information about the argasid machinery involved in blood digestion. This information represents a starting point for the identification and selection of new targets for the development of alternative control strategies.

Experimental Infection of Ornithodoros erraticus sensu stricto with Two Portuguese African Swine Fever Virus Strains. Study of Factors Involved in the Dynamics of Infection in Ticks.

African swine fever (ASF) is a frequently devastating hemorrhagic disease of domestic pigs and wild boar and Ornithodoros erraticus sensu stricto argasid ticks are the only biological vectors of African swine fever virus (ASFV) known to occur in Europe. Recently this disease emerged in Eastern Europe and Russian Federation, showing a huge potential for a rapid spread between countries. There is some risk of re-emergence of ASF in the countries where these ticks exist, that can contribute for the persistence of infection and compromise control measures. In this study we aimed to identify factors that determine the probability of infection and its dynamics in the tick vector Ornithodoros erraticus sensu stricto, with two Portuguese strains of ASFV. Our results suggest that these ticks have a high likelihood of excreting the two haemadsorbing ASF viruses of different host origins and that, in field surveys, the analysis of adults and 5th nymphal stage can provide the best chance of detecting virus infection. The results also indicate that infection of pigs with highly virulent ASF viruses will promote higher rates of infection and a higher likelihood for virus excretion by ticks. Nevertheless, there is also a risk, although lower, that ticks can become infected on pigs that have overcome the acute phase of infection, which was simulated in our study by membrane feeding ticks with low titres of virus. We believe these results can be valuable in designing and interpreting the results of ASF control programmes, and future work can also be undertaken as our dataset is released under open access, to perform studies in risk assessment for ASFV persistence in a region where O. erraticus sensu stricto ticks are present.

New salivary anti-haemostatics containing protective epitopes from Ornithodoros moubata ticks: Assessment of their individual and combined vaccine efficacy.

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 A2 (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.

Identification and comparative analysis of subolesin/akirin ortholog from Ornithodoros turicata ticks.

BACKGROUND: Subolesin is an evolutionary conserved molecule in diverse arthropod species that play an important role in the regulation of genes involved in immune responses, blood digestion, reproduction and development. In this study, we have identified a subolesin ortholog from soft ticks Ornithodoros turicata, the vector of the relapsing fever spirochete in the United States. METHODS: Uninfected fed or unfed O. turicata ticks were used throughout this study. The subolesin mRNA was amplified by reverse transcription polymerase chain reaction (RT-PCR) and sequenced. Quantitative-real time PCR (QRT-PCR) was performed to evaluate subolesin mRNA levels at different O. turicata developmental stages and from salivary glands and gut tissues. Bioinformatics and comparative analysis was performed to predict potential post-translational modifications in O. turicata subolesin amino-acid sequences. RESULTS: Our study reveals that O. turicata subolesin gene expression is developmentally regulated, where; adult ticks expressed significantly higher levels in comparison to the larvae or nymphal ticks. Expression of subolesin was evident in both unfed and fed ticks and in the salivary glands and midgut tissues. The expression of subolesin transcripts varied in fed ticks with peak levels at day 14 post-feeding. Phylogenetic analysis revealed that O. turicata subolesin showed a high degree of sequence conservation with subolesin's from other soft and hard ticks. Bioinformatics and comparative analysis predicted that O. turicata subolesin carry three Protein kinase C and one Casein kinase II phosphorylation sites. However, no myristoylation or glycosylation sites were evident in the O. turicata subolesin sequence. CONCLUSION: Our study provides important insights in recognizing subolesin as a conserved potential candidate for the development of a broad-spectrum anti-vector vaccine to control not only ticks but also several other arthropods that transmit diseases to humans and animals.

Self-assembled protein arrays from an Ornithodoros moubata salivary gland expression library.

Protein interactions play a critical role in the regulation of many biological events and their study in a high-throughput format has become a key area of proteomic research. Nucleid Acid Programmable Protein Arrays (NAPPA) technology allows the construction of protein arrays from cDNA expression libraries in high-throughput cell-free systems to study protein interaction and functions. Tick saliva contains antihemostatic, anti-inflammatory, and immunosuppressive proteins that counteract the host hemostatic, immune, and inflammatory responses allowing the ingestion of host blood and facilitating its infection by the tick-borne pathogens. Identification of such proteins and their functions could help in the selection of antigenic targets for the development of antitick and transmission-blocking vaccines. With that aim, we have prepared a cDNA expression library from the salivary glands of Ornithodoros moubata and subsequently produced a self-assembled protein microarray using 480 randomly selected clones from that library. The reproducibility of the array, its representativeness of the tick salivary protein repertoire, and the functionality of the in situ expressed proteins have been checked, demonstrating that it is a suitable tool for the identification and functional characterization of soft tick salivary molecules that interact with host proteins. Several clones in the array were shown to bind to human recombinant P-selectin. One of them was a likely secreted tick phospholipase A2, which may represent a potential new ligand for P-selectin. As these salivary molecules are likely involved in blood meal acquisition through the modulation of the host immune and hemostatic responses, this new high-throughput tool could open new avenues for development of new therapeutic agents and control strategies against ticks and tick-borne pathogens.

Subolesin/akirin orthologs from Ornithodoros spp. soft ticks: cloning, RNAi gene silencing and protective effect of the recombinant proteins.

Subolesin/akirin is a well characterized protective antigen highly conserved across vector species and thus potentially useful for the development of a broad-spectrum vaccine for the control of arthropod infestations including hard ticks, mosquitoes, sand flies and the poultry red mite Dermanyssus gallinae. Soft ticks could be also targeted by this vaccine if proved that the soft tick subolesin orthologs are conserved and induce protective immune responses too. However, to date no soft tick subolesin orthologs have been fully characterized nor tested as recombinant antigens in vaccination trials. The objectives of the present work were to clone and characterize the subolesin orthologs from two important vector species of soft ticks as Ornithodoros erraticus and O. moubata, to evaluate the effect of subolesin gene silencing by RNAi, and to test the protective value of the recombinant antigens in vaccination trials. The obtained results demonstrate that both soft tick subolesins are highly conserved showing more than 69% and 74% identity with those of hard ticks in their nucleotide and amino acid sequences, respectively. Additionally, we demonstrate that both soft ticks possess fully operative RNAi machinery, and that subolesin gene silencing by dsRNA injection inhibits oviposition indicating the involvement of subolesin in tick reproduction. Finally, vaccination with the recombinant soft tick subolesins induced a partial protective effect resulting in the reduction of the oviposition rate. These preliminary results encourage further studies on the use of recombinant subolesins as vaccines for the control of soft tick infestations, either alone or in combination with other specific molecules.

Cloning, characterization and diagnostic performance of the salivary lipocalin protein TSGP1 from Ornithodoros moubata.

The argasid tick Ornithodoros moubata is distributed throughout South and East Africa and Madagascar, where it colonizes wild and domestic habitats and feeds on warthogs, domestic swine, and humans. This argasid transmits the spirochete Borrelia duttonii, causing East African tick-borne relapsing fever in humans, and the African swine fever virus, which causes a highly lethal haemorrhagic disease in pigs. Tick surveillance and the elimination of O. moubata from synanthropic environments (human dwellings and pigsties) would facilitate the control and prevention of these two diseases. Since direct surveillance methods are impractical in this context, the development of an indirect method for the detection of specific antibodies against tick salivary proteins in samples taken from animal or human hosts living in the area under study would provide a more convenient surveillance and diagnostic tool. Previous work has indicated that the 20A1 salivary antigen of O. moubata could be an optimal candidate for the development of a specific serological test and identified it as an orthologue of the Ornithodoros savignyi TSGP1 lipocalin. The objectives of the present work were to clone, sequence, and molecularly characterize the O. moubata TSGP1, as well as its production as a recombinant protein in order to assess its usefulness as a diagnostic antigen in an ELISA test for tick surveillance. Our results show that O. moubata TSGP1 (OmTSGP1) conserves the tertiary structure of lipocalins and contains the biogenic amine-binding motif. We also show that OmTSGP1 shares 65% sequence identity with the O. savignyi TSGP1, demonstrating that they represent orthologous proteins and suggesting they share identical function as biogenic amine scavengers. A recombinant form of OmTSGP1 was produced, showing 100% sensitivity and 99.4% specificity in an ELISA test for the detection of anti-O. moubata antibodies in pig sera. This recombinant antigen represents a promising epidemiological tool for O. moubata surveillance that may help to implement control measures against O. moubata-borne diseases.

Savicalin, a lipocalin from hemocytes of the soft tick, Ornithodoros savignyi.

Savicalin, is a lipocalin found in the hemocytes of the soft tick, Ornithodoros savignyi. It could be assigned to the tick lipocalin family based on BLAST analysis. Savicalin is the first non-salivary gland lipocalin described in ticks. The mature sequence is composed of 188 amino acids with a molecular mass of 21481.9 Da. A homolog for savicalin was found in a whole body EST-library from a related soft tick O. porcinus, while other tick salivary gland derived lipocalins retrieved from the non-redundant sequence database are more distantly related. Homology modeling supports the inclusion of savicalin into the lipocalin family. The model as well as multiple alignments suggests the presence of five disulphide bonds. Two conserved disulphide bonds are found in hard and soft tick lipocalins. A third disulphide bond is shared with the TSGP4-clade of leukotriene C4 binding soft tick lipocalins and a fourth is shared with a lipocalin from the hard tick Ixodes scapularis. The fifth disulphide bond is unique and links strands D-E. Phylogenetic analysis showed that savicalin is a distant relative of salivary gland derived lipocalins, but groups within a clade that is possibly non-salivary gland derived. It lacks the biogenic amine-binding motif associated with tick histamine and serotonin binding proteins. Expression profiles indicate that savicalin is found in hemocytes, midgut and ovaries, but not in the salivary glands. Up-regulation occurs in hemocytes after bacterial challenge and in midguts and ovaries after feeding. Given its tissue distribution and up-regulation of expression, it is possible that this lipocalin functions in tick development after feeding or in an anti-microbial capacity.

Isolation and expression of the retinoid X receptor from last instar nymphs and adult females of the soft tick Ornithodoros moubata (Acari: Argasidae).

Retinoid X receptors (RXR) exist broadly from invertebrates to vertebrates, and play essential roles in physiological processes of these organisms. In arthropods, RXRs form a complex with the ecdysteroid receptor (EcR) and ecdysteroids to mediate the regulation of ecdysis and reproduction. Compared to EcR, RXR and its homologue ultraspiracle (USP) are much less well understood. Therefore, we identified RXR of the soft tick Ornithodoros moubata (OmRXR) and used real-time PCR to examine the expression of OmRXR. This is the first report of RXR from a soft tick. OmRXR showed higher homology to hard tick, crustacean and vertebrate RXRs than insect RXRs and USPs. OmRXR expression was observed during molting in the last instar nymphs coinciding with EcR expression and increases in ecdysteroid titers. Tick vitellogenesis normally occurs soon after engorgement and OmRXR expression coinciding with EcR expression and ecdysteroid titers in engorged females occurred before vitellogenin (Vg) synthesis and egg maturation. The ecdysteroid/EcR/RXR complex appears to be important in the regulation of molting and vitellogenesis of soft ticks.

An insight into the sialome of the soft tick, Ornithodorus parkeri.

While hard ticks (Ixodidae) take several days to feed on their hosts, soft ticks (Argasidae) feed faster, usually taking less than 1h per meal. Saliva assists in the feeding process by providing a cocktail of anti-hemostatic, anti-inflammatory and immunomodullatory compounds. Saliva of hard ticks has been shown to contain several families of genes each having multiple members, while those of soft ticks are relatively unexplored. Analysis of the salivary transcriptome of the soft tick Ornithodorus parkeri, the vector of the relapsing fever agent Borrelia parkeri, indicates that gene duplication events have led to a large expansion of the lipocalin family, as well as of several genes containing Kunitz domains indicative of serine protease inhibitors, and several other gene families also found in hard ticks. Novel protein families with sequence homology to insulin growth factor-binding protein (prostacyclin-stimulating factor), adrenomedulin, serum amyloid A protein precursor and similar to HIV envelope protein were also characterized for the first time in the salivary gland of a blood-sucking arthropod. The sialotranscriptome of O. parkeri confirms that gene duplication events are an important driving force in the creation of salivary cocktails of blood-feeding arthropods, as was observed with hard ticks and mosquitoes. Most of the genes coding for expanded families are homologous to those found in hard ticks, indicating a strong common evolutionary path between the two families. As happens to all genera of blood-sucking arthropods, several new proteins were also found, indicating the process of adaptation to blood feeding still continues to recent times.