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Ticks are blood-feeding arthropod ectoparasites of wild and domestic animals that transmit disease-causing pathogens to humans and animals worldwide and a good model for the characterization of tick-host-pathogen interactions. Tick-host-pathogen interactions consist of dynamic processes involving genetic traits of hosts, pathogens, and ticks that mediate their development and survival. Proteomics provides information on the protein content of cells and tissues that may differ from results at the transcriptomics level and may be relevant for basic biological studies and vaccine antigen discovery. In this chapter, we describe various methods for protein extraction and for proteomics analysis in ticks based on one-dimensional gel electrophoresis to characterize tick-host-pathogen interactions. Particularly relevant for this characterization is the use of blood-fed ticks. Therefore, we put special emphasis on working with replete ticks collected after feeding on vertebrate hosts.

[Background]: Ixodid ticks are important vectors of a wide variety of viral, bacterial and protozoan pathogens of medical and veterinary importance. Although several studies have elucidated tick responses to bacteria, little is known about the tick response to viruses. To gain insight into the response of tick cells to flavivirus infection, the transcriptomes and proteomes of two Ixodes spp cell lines infected with the flavivirus tick-borne encephalitis virus (TBEV) were analysed. [Methods]: RNA and proteins were isolated from the Ixodes scapularis-derived cell line IDE8 and the Ixodes ricinus-derived cell line IRE/CTVM19, mock-infected or infected with TBEV, on day 2 post-infection (p.i.) when virus production was increasing, and on day 6 p.i. when virus production was decreasing. RNA-Seq and mass spectrometric technologies were used to identify changes in abundance of, respectively, transcripts and proteins. Functional analyses were conducted on selected transcripts using RNA interference (RNAi) for gene knockdown in tick cells infected with the closely-related but less pathogenic flavivirus Langat virus (LGTV). [Results]: Differential expression analysis using DESeq resulted in totals of 43 and 83 statistically significantly differentially-expressed transcripts in IDE8 and IRE/CTVM19 cells, respectively. Mass spectrometry detected 76 and 129 statistically significantly differentially-represented proteins in IDE8 and IRE/CTVM19 cells, respectively. Differentially-expressed transcripts and differentially-represented proteins included some that may be involved in innate immune and cell stress responses. Knockdown of the heat-shock proteins HSP90, HSP70 and gp96, the complement-associated protein Factor H and the protease trypsin resulted in increased LGTV replication and production in at least one tick cell line, indicating a possible antiviral role for these proteins. Knockdown of RNAi-associated proteins Argonaute and Dicer, which were included as positive control

Anaplasma marginale is an economically important tick-borne pathogen of cattle that causes bovine anaplasmosis. A wide range of geographic strains of A. marginale have been isolated from cattle, several of which have been characterized using genomics and proteomics. While many of these strains have been propagated in tick lines, comparative analyses after propagation in tick cells have not been reported. The overall purpose of this research therefore was to compare the degree of conservation of selected genes after propagation in tick cell culture among A. marginale strains from the U.S. (the Virginia strain) and Brazil (UFMG1 and UFMG2 strains). The genes studied herein included those which encode the proteins HSP70 and SODB involved in heat shock and stress responses, respectively, and two genes that encode major surface proteins MSP4 and MSP5. Strain identities were first confirmed by sequencing the tandem repeats of the msp1a gene which encodes for the adhesin, MSP1a. The results of these studies demonstrated that the genes encoding for both stress response and heat shock proteins were highly conserved among the three A. marginale strains. Antibodies specific for MSP4, MSP5, SODB and HSP70 proteins were used to further characterize the A. marginale strains, and they reacted with all of these strains propagated in tick cell culture, providing further evidence for antigenic conservation. Although antigenic differences were not found among the three A. marginale strains, multi-locus sequence analysis (MLSA) performed with nucleotide sequences of these genes demonstrated that the A. marginale Brazilian and U.S. strains fall in different clades. These results showed that phylogenetically distant strains of A. marginale are antigenically conserved, even after several in vitro passages, supporting the use of some of the above conserved proteins as candidates for universal vaccines.

Ticks transmit zoonotic pathogens worldwide. Nevertheless, very little information is available on their genome, transcriptome and proteome. Herein, we characterized the proteome of Amblyomma americanum adults and nymphs because of their role in pathogen transmission and compared the proteome of A. americanum, A. cajennense and A. variegatum adult ticks. We also used de novo sequencing proteomics data for the analysis of the phylogenetic relationships between the three Amblyomma spp. in a proof of concept for phyloproteomics. The results showed that host and tick proteins involved in blood digestion, heme detoxification, development and innate immunity were differentially represented between adults and nymphs. Although these ticks were unfed, over-represented host proteins may supply nutrients during off-host periods. Tick proteins involved in tick attachment, feeding, heat shock response, protease inhibition and heme detoxification were differentially represented between Amblyomma spp., suggesting adaptation processes to biotic and abiotic factors. These results suggested that phyloproteomics might be a useful tool for the phylogenetic analysis of tick species in which sequence data is a limiting factor and demonstrate the possibilities of proteomics studies for the characterization of relevant tick vector species and provide new relevant information to understand the physiology, development and evolution of these tick species. [Biological significance]: This is the first report on the proteome of the most important Amblyomma tick species for their relevance as vectors of zoonotic pathogens worldwide. Nevertheless, very little information is available on the genome, transcriptome and proteome of these vector ectoparasites. The results reported herein provide new relevant information to understand the physiology, development and evolution of these tick species. Phyloproteomics using de novo protein sequencing was assayed as a new approach for the phylogenetic analys

[Background]: Dermacentor reticulatus (Fabricius, 1794) is distributed in Europe and Asia where it infests and transmits disease-causing pathogens to humans, pets and other domestic and wild animals. However, despite its role as a vector of emerging or re-emerging diseases, very little information is available on the genome, transcriptome and proteome of D. reticulatus. Tick larvae are the first developmental stage to infest hosts, acquire infection and transmit pathogens that are transovarially transmitted and are exposed to extremely stressing conditions. In this study, we used a systems biology approach to get an insight into the mechanisms active in D. reticulatus unfed larvae, with special emphasis on stress response. [Principal Findings]: The results support the use of paired end RNA sequencing and proteomics informed by transcriptomics (PIT) for the analysis of transcriptomics and proteomics data, particularly for organisms such as D. reticulatus with little sequence information available. The results showed that metabolic and cellular processes involved in protein synthesis were the most active in D. reticulatus unfed larvae, suggesting that ticks are very active during this life stage. The stress response was activated in D. reticulatus unfed larvae and a Rickettsia sp. similar to R. raoultii was identified in these ticks. [Significance]: The activation of stress responses in D. reticulatus unfed larvae likely counteracts the negative effect of temperature and other stress conditions such as Rickettsia infection and favors tick adaptation to environmental conditions to increase tick survival. These results show mechanisms that have evolved in D. reticulatus ticks to survive under stress conditions and suggest that these mechanisms are conserved across hard tick species. Targeting some of these proteins by vaccination may increase tick susceptibility to natural stress conditions, which in turn reduce tick survival and reproduction, thus reducing tick populat

Infestations with cattle ticks, Rhipicephalus (Boophilus) microplus and Rhipicephalus annulatus, economically impact cattle production in tropical and subtropical regions of the world. Vaccines containing the recombinant R. microplus BM86 gut antigen were developed and commercialized to induce an immunological protection in cattle against tick infestations. These vaccines demonstrated that tick control by vaccination is cost-effective, reduces environmental contamination and prevents the selection of drug resistant ticks that result from repeated acaricide applications. The protection elicited by BM86-containing vaccines against tick infestations is mediated by a collaborative action between the complement system and IgG antibodies. The efficacy of the vaccination with BM86 and other tick antigens is always higher for R. annulatus than against R. microplus, suggesting that tick genetic and/or physiological factors may affect tick vaccine efficacy. These factors may be related to BM86 protein levels or tick physiological processes such as feeding and protein degradation that could result in more efficient antibody-antigen interactions and vaccine efficacy. To test this hypothesis, we compared the proteome in R. annulatus and R. microplus female ticks after feeding on BM86-vaccinated and control cattle. The results showed that cattle proteins were under represented in R. annulatus when compared to R. microplus, suggesting that R. annulatus ticks ingested less blood, a difference that increased when feeding on vaccinated cattle, probably reflecting the effect of antibody-BM86 interactions on this process. The results also showed that tick protein degradation machinery was under represented in R. annulatus when compared to R. microplus. BM86 mRNA and protein levels were similar in both tick species, suggesting that lesser protease activity in R. annulatus results in more efficient antibody-antigen interactions and higher vaccine efficacy. These results have important im

Cattle ticks, Rhipicephalus (Boophilus) microplus, are a serious threat to animal health and production. Some ticks feed on a single host species while others such as R. microplus infest multiple hosts. White tailed deer (WTD) play a role in the maintenance and expansion of cattle tick populations. However, cattle ticks fed on WTD show lower weight and reproductive performance when compared to ticks fed on cattle, suggesting the existence of host factors that affect tick feeding and reproduction. To elucidate these factors, a proteomics approach was used to characterize tick and host proteins in R. microplus ticks fed on cattle and WTD. The results showed that R. microplus ticks fed on cattle have overrepresented tick proteins involved in blood digestion and reproduction when compared to ticks fed on WTD, while host proteins were differentially represented in ticks fed on cattle or WTD. Although a direct connection cannot be made between differentially represented tick and host proteins, these results suggested that differentially represented host proteins together with other host factors could be associated with higher R. microplus tick feeding and reproduction observed in ticks fed on cattle.

Ticks are vectors of pathogens that affect human and animal health worldwide. Ticks and the pathogens they transmit have co-evolved molecular interactions involving genetic traits of both the tick and the pathogen that mediate their development and survival. Proteomics and genomics studies of infected ticks are required to understand tick-pathogen interactions and identify potential vaccine antigens to control tick infestations and pathogen transmission. In this paper, the application of quantitative proteomics to characterize differential protein expression in ticks and cultured tick cells in response to pathogen infection is reviewed. Analyses using (a) two-dimensional differential in gel electrophoresis (DIGE) labeling and (b) protein one-step in gel digestion, peptide iTRAQ labeling, and isoelectric focusing fractionation, both followed by peptide and protein identifications by mass spectrometry resulted in the identification of host, pathogen, and tick proteins differentially expressed in response to infection. Although at its infancy, these results showed that quantitative proteomics is a powerful approach to characterize the tick-pathogen interface and demonstrated pathogen and tick-specific differences in protein expression in ticks and cultured tick cells in response to pathogen infection.