The mechanisms regulating exocytosis of the salivary glands of the soft tick,Ornithodoros savignyi

Numerous bioactive compounds are secreted from large dense core granules in tick salivary glands during feeding. Investigations into the signalling pathways regulating secretion indicated that they are similar for Argasidae (fast feeding ticks) and Ixodidae (slow-feeding ticks). In both cases, dopamine is the external signal that activates adenylyl cyclase, subsequently cyclic AMP levels are increased and Protein Kinase A (PKA) is activated, resulting in the phosphorylation of proteins. Secretion was also found to be highly calcium dependant. Firstly, it requires extracellular calcium (via a L-type voltage-gated calcium channel located on the plasma membrane) and secondly, intracellular calcium which is released presumably in response to IP3. In contrast to numerous exocrine cells, membrane depolarisation and elevation of the cAMP levels are not sufficient for inducing exocytosis from O. savignyi salivary glands. Pathways such as the activation of Phospholipase C, inositol-phosphate kinases, Na+K+-ATPases, as well as the disassembly of the actin barrier, have been shown to be essential. Finally, our research also indicated a need for the ATPase NSF, an intact microtubule network and an active cytosolic Phospholipase A2 for exocytosis. A model has been suggested, but a great deal of research is needed to elucidate all the mechanisms of regulated exocytosis. All secretory eukaryotic cells to date require SNARE proteins for fusion of granules with the plasma membrane, leading to the release of granular content. By means of Western blotting we identified the tick homologues of the SNAREs syntaxin, SNAP25 and VAMP, as well as the small GTPase Rab3a, all enriched within the membrane fraction. We also identified the SDS-resistant 20S complex, which forms during the docking of granules and is composed of the three SNARE proteins. Confocal microscopy of the SNARE proteins indicates SNAP25 and VAMP localize to the granule membranes, while syntaxin localises strictly to the plasma membrane. In order to isolate the tick SNARE homologues we exploited protein-protein interactions by means of the yeast two-hybrid system. Screening of an O. savignyi cDNA salivary gland library using rat brain a-SNAP as bait, we identified a transcript encoding a tick syntaxin homologue. It encoded a 126 residue protein which shares 14% identity and 40% similarity with human syntaxin 1. Furthermore, we were able to successfully model the identified protein onto the known crystal structure of human syntaxin 1 and indicate that it shares structural homology with helices 1, 2, 3 and the connecting two loop regions. Following screening of the library with a truncated syntaxin bait construct, two novel domains were identified in all the interacting clones. To date their identity remains unknown. Functional complementation in the syntaxin knockout yeast strain H603 with an O. savignyi cDNA library resulted in the identification of four novel transcripts, which suppressed the temperature sensitive phenotype. Two of these share homology with the N- and C-terminals of syntaxins respectively and were successfully modelled onto the human syntaxin 1 crystal structure. Finally, by exploiting the extensive SNARE binding properties of recombinant rat brain a-SNAP, we were able to isolate the O. savignyi SNAREs, i.e. syntaxin, SNAP25 and VAMP, using pull-down assays. These purified proteins will soon be subjected to amino acid sequencing, and their sequences used to confirm the identified transcripts as true syntaxins. By enhancing our understanding of the molecular basis underlying tick feeding, as well as the proteins involved in the processes, we hope to identify possible targets for the rational design of a viable tick vaccine.Thesis (PhD (Biochemistry))--University of Pretoria, 2007.Biochemistryunrestricte

Insight into the Machinery and Applications for Understanding the Pathogen- Vector Interface

The availability of genome sequencing data in combination with knowledge of expressed genes via transcriptome and proteome data has greatly advanced our understanding of arthropod vectors of disease. Not only have we gained insight into vector biology, but also into their respective vector-pathogen interactions. By combining the strengths of postgenomic databases and reverse genetic approaches such as RNAi, the numbers of available drug and vaccine targets, as well as number of transgenes for subsequent transgenic or paratransgenic approaches, have expanded. These are now paving the way for in- field control strategies of vectors and their pathogens. Basic scientific questions, such as understanding the basic components of the vector RNAi machinery, is vital, as this allows for the transfer of basic RNAi machinery components into RNAi-deficient vectors, thereby expanding the genetic toolbox of these RNAi-deficient vectors and pathogens. In this review, we focus on the current knowledge of arthropod vector RNAi machinery and the impact of RNAi on understanding vector biology and vector-pathogen interactions for which vector genomic data is available on VectorBase

Comparative microarray analyses of adult female midgut tissues from feeding Rhipicephalus species

The cattle tick, Rhipicephalus microplus, has a debilitating effect on the livestock industry worldwide, owing to its being a vector of the causative agents of bovine babesiosis and anaplasmosis. In South Africa, co-infestation with R. microplus and R. decoloratus, a common vector species on local livestock, occurs widely in the northern and eastern parts of the country. An alternative to chemical control methods is sought in the form of a tick vaccine to control these tick species. However, sequence information and transcriptional data for R. decoloratus is currently lacking. Therefore, this study aimed at identifying genes that are shared between midgut tissues of feeding adult female R. microplus and R. decoloratus ticks. In this regard, a custom oligonucleotide microarray comprising of 13,477 R. microplus sequences was used for transcriptional profiling and 2476 genes were found to be shared between these Rhipicephalus species. In addition, 136 transcripts were found to be more abundantly expressed in R. decoloratus and 1084 in R. microplus. Chi-square analysis revealed that genes involved in lipid transport and metabolism are significantly overrepresented in R. microplus and R. decoloratus. This study is the first transcriptional profiling of R. decoloratus and is an additional resource that can be evaluated further in future studies for possible tick control.The Red Meat Research Development Trust Gauteng Department of Agriculture and Rural Development,University of Pretoria Research Development Program, and the National Research Foundation’s Technology and Human Resourcesfor Industry Program (Grant No. TP2010072300035).http://www.elsevier.com/locate/ttbdishb201

A systematic, functional genomics, and reverse vaccinology approach to the identification of vaccine candidates in the cattle tick, Rhipicephalus

In the post-genomic era, reverse vaccinology is proving promising in the development of vaccines against bacterial and viral diseases, with limited application in ectoparasite vaccine design. In this study, we present a systematic approach using a combination of functional genomics (DNA microarrays) techniques and a pipeline incorporating in silico prediction of subcellular localization and protective antigenicity using VaxiJen for the identification of novel anti-tick vaccine candidates. A total of 791 candidates were identified using this approach, of which 176 are membrane-associated and 86 secreted soluble proteins. A preliminary analysis on the antigenicity of selected membrane proteins using anti-gut antisera yielded candidates with an IgG binding capacity greater than previously identified epitopes of Bm86. Subsequent vaccination trials using recombinant proteins will not only validate this approach, but will also improve subsequent reverse vaccinology approaches for the identification of novel anti-tick vaccine candidates.The Red Meat Research Development Trust, University of Pretoria Research Development Programme, and the Technology and Human Resources for Industry Programme.http://www.elsevier.de/ttbdishb201

Transmembrane proteins - mining the cattle tick transcriptome

Managing the spread and load of pathogen-transmitting ticks is an important task worldwide. The cattletick, Rhipicephalus microplus, not only impacts the economy through losses in dairy and meat pro-duction, but also raises concerns for human health in regards to the potential of certain transmittedpathogens becoming zoonotic. However, novel strategies to control R. microplus are hindered by lack ofunderstanding tick biology and the discovery of suitable vaccine or acaricide targets. The importance oftransmembrane proteins as vaccine targets are well known, as is the case in tick vaccines with Bm86 asantigen. In this study, we describe the localization and functional annotation of 878 putative transmem-brane proteins. Thirty proteins could be confirmed in the R. microplus gut using LC-MS/MS analysis andtheir roles in tick biology are discussed. To the best of our knowledge, 19 targets have not been reportedbefore in any proteomics study in various tick species and the possibility of using the identified proteins astargets for tick control are discussed. Although tissue expression of identified putative proteins throughexpansive proteomics is necessary, this study demonstrates the possibility of using bioinformatics forthe identification of targets for further evaluation in tick control strategies.Red Meat Research Development Trust, the University of Pretoria Research Development Programme and the Technology and Human Resources for Industry Programme.http://www.elsevier.com/locate/ttbdis2016-09-30hb201

Gene expression profiling of adult female tissues in feeding Rhipicephalus microplus cattle ticks

The southern cattle tick, Rhipicephalus microplus, is an economically important pest, especially for resource-poor countries, both as a highly adaptive invasive species and prominent vector of disease. The increasing prevalence of resistance to chemical acaricides and variable efficacy of current tick vaccine candidates highlight the need for more effective control methods. In the absence of a fully annotated genome, the wealth of available expressed sequence tag sequence data for this species presents a unique opportunity to study the genes that are expressed in tissues involved in blood meal acquisition, digestion and reproduction during feeding. Utilising a custom oligonucleotide microarray designed from available singletons (BmiGI Version 2.1) and expressed sequence tag sequences of R. microplus, the expression profiles in feeding adult female midgut, salivary glands and ovarian tissues were compared. From 13,456 assembled transcripts, 588 genes expressed in all three tissues were identified from fed adult females 20 days post infestation. The greatest complement of genes relate to translation and protein turnover. Additionally, a number of unique transcripts were identified for each tissue that relate well to their respective physiological/biological function/role(s). These transcripts include secreted anti-hemostatics and defense proteins from the salivary glands for acquisition of a blood meal, proteases as well as enzymes and transporters for digestion and nutrient acquisition from ingested blood in the midgut, and finally proteins and associated factors involved in DNA replication and cell-cycle control for oogenesis in the ovaries. Comparative analyses of adult female tissues during feeding enabled the identification of a catalogue of transcripts that may be essential for successful feeding and reproduction in the cattle tick, R. microplus. Future studies will increase our understanding of basic tick biology, allowing the identification of shared proteins/pathways among different tissues that may offer novel targets for the development of new tick control strategies.The Red Meat Research Development Trust, University of Pretoria Research Development Programme and the Technology and Human Resources for Industry Programme, South Africa. Additional funding and student support was obtained from the National Research Foundation of South Africa.http://www.elsevier.com/locate/ijpar

Probing the Rhipicephalus bursa sialomes in potential anti-tick vaccine candidates : a reverse vaccinology approach

In the wake of the ‘omics’ explosion of data, reverse vaccinology approaches are being applied more readily as an alternative for the discovery of candidates for next generation diagnostics and vaccines. Promising protective antigens for the control of ticks and tick-borne diseases can be discovered by mining available omics data for immunogenic epitopes. The present study aims to explore the previously obtained Rhipicephalus bursa sialotranscriptome during both feeding and Babesia infection, to select antigenic targets that are either membrane-associated or a secreted protein, as well as unique to the ectoparasite and not present in the mammalian host. Further, they should be capable of stimulating T and B cells for a potential robust immune response, and be non-allergenic or toxic to the host. From the R. bursa transcriptome, 5706 and 3025 proteins were identified as belonging to the surfaceome and secretome, respectively. Following a reverse genetics immunoinformatics pipeline, nine preferred candidates, consisting of one transmembrane-related and eight secreted proteins, were identified. These candidates showed a higher predicted antigenicity than the Bm86 antigen, with no homology to mammalian hosts and exposed regions. Only four were functionally annotated and selected for further in silico analysis, which examined their protein structure, surface accessibility, flexibility, hydrophobicity, and putative linear B and T-cell epitopes. Regions with overlapping coincident epitopes groups (CEGs) were evaluated to select peptides that were further analyzed for their physicochemical characteristics, potential allergenicity, toxicity, solubility, and potential propensity for crystallization. Following these procedures, a set of three peptides from the three R. bursa proteins were selected. In silico results indicate that the designed epitopes could stimulate a protective and long-lasting immune response against those tick proteins, reflecting its potential as anti-tick vaccines The immunogenicity of these peptides was evaluated in a pilot immunization study followed by tick feeding to evaluate its impact on tick behavior and pathogen transmission. Combining in silico methods with in vivo immunogenicity evaluation enabled the screening of vaccine candidates prior to expensive infestation studies on the definitive ovine host animals.Spreadsheet S1 – SurfaceomeSpreadsheet S2 – SecretomeSpreadsheet S3 – MARVELSpreadsheet S4 – EVASINSpreadsheet S5 - RICINFundação para a Ciência e Tecnologia (FCT)http://www.mdpi.com/journal/biomedicinespm2021BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant PathologyPlant Production and Soil Scienc

SNP analysis infers that recombination is involved in the evolution of Amitraz resistance in Rhipicephalus microplus

Rhipicephalus microplus, better known as the Asiatic cattle tick, is a largely invasive ectoparasite of great economic importance due to the negative effect it has on agricultural livestock on a global scale, particularly cattle. Tick-borne diseases (babesiosis and anaplasmosis) transmitted by R. microplus are alarming as they decrease the quality of livestock health and production. In sub-Saharan Africa, cattle represent a major source of meat and milk, but this region of the world is severely affected by the Rhipicephalus microplus tick. The principal method for tick control is the use of chemical acaricides, notably amitraz, which was implemented in the 1990’s after resistance to other acaricides surfaced. However, the efficiency of chemical control is hindered by an increase in the frequency of mutant resistance alleles to amitraz in tick populations. Presently, the only way to assess amitraz resistance is by means of larval packet tests, but this technique is time-consuming and not particularly cost effective. The main aims of this study were three-fold. First, we attempted to correlate two known SNPs in the octopamine/tyramine (OCT/Tyr) receptor with amitraz resistance in South African field samples of R. microplus. Second, we calculated gametic disequilibrium for these SNPs to determine whether they are randomly associated. Lastly, we conducted a study to assess the evolutionary effects of recombination within the OCT/ Tyr receptor. Our results confirmed that the two SNPs are associated with amitraz resistance in the South African tick strain, and that they are in gametic disequilibrium. Additionally, recombination was detected in the OCT/Tyr receptor generating two recombinant haplotypes. These results are of concern to farmers in sub-Saharan Africa, and the emergence of amitraz resistance should be closely monitored in future. Therefore, we present a quick and affordable RFLP based diagnostic technique to assess amitraz resistance in field samples of R. microplus.S1 Fig. Subpopulation structure of ticks across South Africa. Ticks from each farm were placed into subpopulations (1–15) depending on the region from which they were collected. Grid blocks were constructed 300 x 300 km over the country for accurate overall segregation of populations. The farms from which tick samples were analyzed are indicated in the table, along with their grid block number and province. Farm numbers correspond with sample number, e.g. sample 44.1MF is sample 1 of female R. microplus from farm 44.S1 Table. GenBank accession numbers for all R. microplus OCT/Tyr receptor sequences.S2 Table. Genotypes of field samples of R. microplus ticks at the two published SNP positions.S3 Table. Rhipicephalus microplus larval packet test results.Funding was provided by (a) Gauteng Department of Agriculture and Rural Development, C Maritz-Olivier. (b) Zoetis South Africa (Pty) Ltd., C Maritz-Olivier (c) National Research Foundation, THRIP grant nr: 83890.http://www.plosone.orgam201

Evolution of the tissue factor pathway inhibitor-like Kunitz domain-containing protein family in Rhipicephalus microplus

One of the principle mechanisms utilised by ticks to obtain a blood meal is the subversion of the host’s haemostatic response. This is achieved through the secretion of saliva containing anti-haemostatic proteins into the feeding lesion. Lineage-specific expansion of predicted secretory protein families have been observed in all previously studied ticks and occurred in response to adaptation to a blood-feeding environment. Of these, the predominant families are common between both hard and soft ticks. One of these families, namely the Kunitz domain-containing protein family, includes proven tissue factor pathway inhibitor-like (TFPI-like) anti-haemostatics such as ixolaris and penthalaris that play a crucial role during tick feeding. Although Kunitz-type proteins have been found in Rhipicephalus microplus, the TFPI-like Kunitz protein family has not yet been studied. We report a comprehensive search for TFPI-like Kunitz domain-containing proteins in R. microplus expressed sequence tag libraries, resulting in the identification of 42 homologues. The homologues were bioinformatically and phylogenetically studied, including the application of an intensive Bayesian Markov Chain Monte Carlo (MCMC) analysis of the individual Kunitz domain nucleotide sequences. We show that the R. microplus TFPI-like Kunitz protein family groups into two main clades that presumably underwent ancient duplication, which indicates that a whole genome duplication event occurred at least 150 million years ago. Evidence for recent and ancient gene and domain duplication events was also found. Furthermore, the divergence times of the various tick lineages estimated in this paper correspond with those presented in previous studies. The elucidation of this large protein family’s evolution within R. microplus adds to current knowledge of this economically important tick.This project was partially funded by the Wellcome Trust, United Kingdom, under the ‘Animal Health in the Developing World’ initiative through Project 0757990 entitled ‘Adapting recombinant anti-tick vaccines to livestock in Africa’.http://www.elsevier.com/locate/ijpar

Genetic diversity, acaricide resistance status and evolutionary potential of a Rhipicephalus microplus population from a disease-controlled cattle farming area in South Africa

The Southern cattle tick, Rhipicephalus microplus is a hematophagous ectoparasite of great veterinary and economic importance. Along with its adaptability, reproductive success and vectoring capacity, R. microplus has been reported to develop resistance to the major chemical classes of acaricides currently in use. In South Africa, the Mnisi community in the Mpumalanga region offers a unique opportunity to study the adaptive potential of R. microplus. The aims of this study therefore included characterising acaricide resistance and determining the level and pattern of genetic diversity for R. microplus in this region from one primary population consisting of 12 communal dip-stations. The level of acaricide resistance was evaluated using single nucleotide polymorphisms (SNPs) in genes that contribute to acaricide insensitivity. Additionally, the ribosomal internal transcribed spacer 2 (ITS2) gene fragments of collected individuals were sequenced and a haplotype network was constructed. A high prevalence of alleles attributed to resistance against formamidines (amitraz) in the octopamine/tyramine (OCT/Tyr) receptor (frequency of 0.55) and pyrethroids in the carboxylesterase (frequency of 0.81) genes were observed. Overall, the sampled tick population was homozygous resistant to pyrethroid-based acaricides in the voltage-gated sodium channel (VGS) gene. A total of 11 haplotypes were identified in the Mnisi R. microplus population from ITS2 analysis with no clear population structure. From these allele frequencies it appears that formamidine resistance in the Mnisi community is on the rise, as the R. microplus populations is acquiring or generating these resistance alleles. Apart from rearing multi-resistant ticks to commonly used acaricides in this community these ticks may pose future problems to its surrounding areas.Zoetis (Pty) Ltd., South Africa, the National Research Foundation Technology and Human Resources for Industry Programme (Grant number TP12082911252) and the Belgium Development Cooperation (DGD) FA3 project.http://www.elsevier.com/locate/ttbdis2017-06-30hb2016GeneticsVeterinary Tropical Disease