Exploring the Transcriptomic Data of the Australian Paralysis Tick, Ixodes Holocyclus

GSTF Journal of Veterinary Science (JVet) Vol.3 No.1, 2016

Exploring the transcriptomic data of the Australian paralysis tick, Ixodes holocyclus

Ong CT1, Rodriguez-Valle M1*, Moolhuijzen PM2, Barrero RA2, Hunter A2, Szabo T2, Bellgard MI2, Lew-Tabor AE1,2*

1The University of Queensland, Queensland Alliance for Agriculture & Food Innovation, St. Lucia, 4072, Queensland Australia; 2Murdoch University, Centre for Comparative Genomics, Murdoch, 6150, WA, Australia *Corresponding authors: [email protected]; [email protected]

Abstract - Ixodes holocyclus is the paralysis tick I. holocyclus is a native and unique commonly found in Australia. I. holocyclus does paralysis tick species of Australia that is commonly not cause paralysis in the primary host – found in areas of high humidity and moderate bandicoots, but markedly affects secondary temperature along the east coast of Australia [3, 4]. hosts such as companion animals, livestock and The primary host of I. holocyclus are native humans. Holocyclotoxins are the neurotoxin marsupials such as bandicoots, whereas livestock, molecules in I. holocyclus responsible for companion animals and humans are considered as paralysis symptoms. There is a limited secondary hosts. Figure 1 describes the lifecycles understanding of holocyclotoxins due to the of the three-host tick, I. holocyclus, which is difficulties in purifying and expressing these conformed by four lifecycle stages from egg, larva, toxins in vitro. Next-generation sequencing nymph to adult. As for other Ixodidae (hard ticks), technologies were utilised for the first time to I. holocyclus can feed on hosts for a minimum few generate transcriptome data from two cDNA days up to several weeks [4, 5]. samples –salivary glands samples collected from female adult ticks engorged on paralysed I. holocyclus is also a vector of various companion animals and on bandicoots. Contig- diseases, for example Rickettsial Spotted Fever, encoded proteins in each library were annotated and is able to transmit pathogens to hosts during according to their best BLAST match against infestation [6, 7]. I. holocyclus is capable of several databases and functionally assigned into inducing paralysis in hosts upon engorgement, six protein categories: housekeeping, which eventually leads to death if left untreated [8]. transposable elements, pathogen-related, In addition, I. holocyclus is recognised as the most hypothetical, secreted and novel. The “secreted severe and harmful form of tick paralysis compared protein” category is comprised of ten protein to its counterparts such as Dermacenor andersoni families: enzymes, protease inhibitors, antigens, and Dermacentor variabilis in North America and mucins, immunity-related, lipocalins, glycine- Rhipicephalus evertsi evertsi in Africa [6]. In rich, putative secreted, salivary and toxin-like. contrast, paralysis symptoms caused by Comparisons of contig representation between I. holocyclus infestations are often irreversible and the two libraries reveal the differential deaths occur regardless of the immediate treatment expression of tick proteins collected from given [8]. Between 1900 and 1945, there were different hosts. This study provides a approximately 30 fatal human cases reported preliminary description of the I. holocyclus tick caused by I. holocyclus, victims were mostly under salivary gland transcriptome. 4 years of age [5, 9]. Domestic animals are the major tick paralysis victims with an estimated I. INTRODUCTION number of 100,000 animals affected by the Australian tick paralysis each year [4, 10]. Five to A. Ixodes holocyclus ten percent of affected canines presenting to veterinary clinics reported fatal regardless of tick Ticks belong to the Class Arachnida, removal and antivenom administration [11, 12]. which includes other arthropods such as spiders and scorpions. Ticks are members of the Subclass The development of paralysis syndrome is Acari that consists of three families: Argasidae, dependent on the duration of infestation and the Ixodidae and Nutalliellidae [1]. Ixodes holocyclus composition of the salivary gland (SG). Generally, is a tick species categorized in the family Ixodidae it takes 3-5 days for clinical symptoms to appear in comprised of hard-bodied ticks. In addition, they victims of I. holocyclus. The clinical presentation are referred to as one of the most ancient terrestrial includes loss of appetite, voice and coordination. arachnids and possibly the earliest organisms to Other severe indications are ascending flaccid have evolved with blood-feeding capabilities [2]. paralysis, excessive salivation, asymmetric pupillary dilation and respiratory distress [13]. DOI: 10.5176/2345-7880_3.1.14

Figure 1 – The lifecycle of the three-host tick Ixodes holocyclus [5].

B. Tick saliva and sialome studies Two cDNA libraries were constructed in this study with large numbers of transcripts yielded The saliva of blood-feeding ticks play an from feeding female adult I. holocyclus ticks essential role for effective and successful feeding. engorged on different hosts. This research paper A large array of saliva components are known to aimed to provide a preliminary description of facilitate tick-host interactions and counteract the the transcriptomes of the Australian paralysis hosts’ defences [14, 15]. To provide a tick, I. holocyclus by annotating the contigs comprehensive understanding of tick saliva, next- obtained from the two cDNA libraries and to generation sequencing, which generates high- understand the differential expression of I. throughput sequencing data and deep coverage of holocyclus protein families when engorged on transcriptomes [16], has been used to generate the different hosts. salivary gland transcriptome (sialome) of several genera within ticks [17-20]. Sialome studies II. MATERIALS AND METHODS revealed that different tick species have a set of novel salivary proteins or protein families, which A. Two cDNA samples are unique and beneficial to their own hematophagy (blood-feeding behaviour) [21, 22]. Two pooled RNA samples were prepared These novel salivary proteins are for example anti- from feeding female adult ticks for cDNA library haemostatic, anti-inflammatory and immune- construction and sequencing. Fully engorged modulatory components, which facilitate the tick female adult I. holocyclus were collected from 2 hematophagy [15]. Previous studies on Ixodes groups of hosts – (i) paralysed cats and dogs, and scapularis and Rhipicephalus appendiculatus (ii) bandicoots. The first sample (SG_FA_CD) and suggested that the variation among tick sialomes is second sample (SG_FA_BA) were SG RNA influenced by tick feeding conditions such as the obtained from female adult ticks collected from vertebrate host(s) and/or developmental stages [23, cats and dogs with paralysis symptoms and 24]. However only 3 sialomes, out of the 243 tick bandicoots, respectively. species in the genus of Ixodes, have been described to date including I. scapularis [20, 24], Ixodes pacificus [25] and Ixodes ricinus [17, 26].

III. RESULTS

A. Next generation sequencing and sequencing B. Next generation sequencing assembly

The cDNA libraries were sent to the A total of 130,071,262 reads were Australian Genome Research Facility Ltd (AGRF) generated for SG_FA_CD using Illumina for next generation sequencing. The SG_FA_CD technology. To improve the read quality, Illumina cDNA sample was sequenced using Illumina HiSeq. read quality was filtered using FastQC and The Illumina sequenced reads were then assessed ConDenTri which filtered out 19,968,632 poor with FastQC [27] to avoid inherent biases quality reads and 58,020,916 redundant reads, associated with sequencing and the starting resulting in 26,040,857 paired end reads libraries. In order to standardize the sequencing (52,081,714 reads) for the SG_FA_CD sample. The reads and to eliminate sequencing errors prior to de assembly of Illumina paired-end reads yielded novo assembly, the processed Illumina reads were 41,797 contigs with an average consensus size of subjected to Content Dependent Read Trimmer 830bp for the SG_FA_CD library. Alternatively, (ConDeTri) [28], which removes redundant and 454 pyrosequencing generated 286,047 reads with low scoring reads. Assemblies of Illumina an average read length of 310bp for SG_FA_BA. sequences were performed using Velvet short read SG_FA_CD 454 pyrosequencing reads were assembler [29] for each odd kmer ranged between assembled into 3,772 with an average consensus 59-79, then merged using Velvet’s Oases [30]. The size of 663bp. second SG_FA_BA sample was sequenced using 454 FLX Roche technologies. The 454 B. Functional annotation pyrosequences were assembled with GS Assembler (Newbler) v2.5.3 [31], and resulting final contig All contigs from the two libraries were assemblies were examined in this study. annotated and can be viewed in Supplementary files 1 and 2. Column A contains a specific C. Contig annotation and contig representation in identification code for each contig. The each library descriptions returned from different BLAST searches against various databases are summarized The contigs of the assembled ESTs in column D. Columns E and F describe hits (Expressed Sequence Tag) in the four individual returned from NR and the NR score. Columns G-J libraries were blast using different BLAST searches contain hits returned with Uniprot and the Uniprot (blastx, balstn, or rpblast searches) against various alignment score, Expected-value E, and sequence databases, including non-redundant protein similarity. While columns K-P describe Enzyme database (NR) of the National Centre for commission number, KOG function, KOG general Biotechnology Information (NCBI) [32], the Swiss- categories, KEGG pathway ID, KEGG pathways, Prot database [33], gene ontology (GO) fasta subset KEGG general category, followed by hits returned [34], the NCBI conserved domains database [35], from SMART, PFAM ISU, SSU, SignalP and KOG [36], KEGG [37], PFAM [38] and SMART TMHMM. [39] motifs for contig annotation. All BLAST searches were undertaken using Yabi [40] with the Based on the best matches, each protein- default parameters. encoded contig, for each I. holocyclus library, was functionally annotated in the Excel spreadsheet Transcripts, which have no significant hit using the classification of Schwarz et al [33]. All with any records in different databases, were contigs were functionally classified into 15 main submitted to the SignalP server [41] to identify protein families (column C) describing their possible secreted proteins and the TMHMM server predicted functions – housekeeping, hypothetical, [42] to detect possible trans-membrane helices. pathogen-related, transposable elements, novel, Finally, all transcripts in each individual library putative secreted, saliva, mucin, glycine rich, were functionally classified by the key words enzyme, lipocalin, protease inhibitor, toxin like, provided from the BLAST match, considering their immunity related and antigen. The nineteen protein significant hits from different search engines. All families were conceptually grouped into 6 annotated contigs for each individual library are categories (column D), which are housekeeping compiled in a hyperlinked Excel spreadsheet. The products, hypothetical proteins, pathogen-related number of contigs per functional group of each sequences, transposable elements, novel products library was calculated for comparison of contig and secreted proteins. representation between the two I. holocyclus libraries.

Housekeeping contigs were proteins Table 5 - Brief descriptions and examples for secreted protein involved in organismal systems, environmental/ family cellular processes and signaling, information Secreted Protein Family Descriptions and Examples storage and processing and metabolism. (References) Hypothetical proteins are proteins labeled Antigen Eg: Antigen 5 [31, 55], Antigen hypothetical and putative in NCBI databases, P23 [56] Enzyme Proteins containing enzyme which are predicted proteins that have not been signatures and a secretion signal well characterized. All ticks used for this or are related to secreted transcriptome study were collected from the field enzymes, including enzymes (including from hosts) and may carry pathogens destined to the ER, the Golgi apparatus or to lysosomes [57]. and/or symbionts. Pathogen-related are associated Eg: Metalloprotease, Apyrase, with several contigs originating from pathogens Chitinase found in the two I. holocyclus libraries. A minor Glycine rich Members of the salivary cement number of contigs were reported to be transposable or extracellular matrix of the SG [57]. Eg: Cuticle, GGY peptides, elements, including both class I (retrotransposons) Collagen-like and class II (transposons) transposable elements, Immunity related Antimicrobial peptides and reverse transcriptases and several transposases. pathogen-recognition proteins Contigs, which did not have significant similarities [58, 59] Eg: Defensin, Lysozyme, with protein or nucleotide sequences on current Microplusin, ML domain, Alpha- macroglobulin, Ficolin databases were classified under novel products and Lipocalin Proteins with versatile structures are predicted to be unique proteins in I. holocyclus. carrying hydrophobic ligands The remaining 10 protein families are classified inside its cavity [60] and are under the category of “Secreted”. Descriptions of abundantly expressed in tick SG [25, 32, 33, 61]. Eg: Histamine secreted protein families, as described in previous binding proteins studies, are summarized in Table 5. Figures 5 and Mucin Serine/threonine-rich proteins 6 depict the classification of the categories and that can be heavily glycosylated protein families in the two I. holocyclus libraries. with N-acetyl galactosamine residues [57]. Protease Inhibitor Eg: Kunitz domains, Serpins, B. 1. Functional annotations for Salivary Gland Cystatins [57, 61] collected from Female Adults collected from Putative Secreted Hypothetical secreted proteins paralysed Cats and Dogs (SG_FA_CD) Protein identified in other ticks [31, 61] Salivary Putative secreted salivary gland peptides identified in other ticks For the SG_FA_CD library, a total [24, 31, 61] number of 21,982 contigs (52.85%) were predicted Toxin like Protein family with six conserved to encode for novel proteins (Figure 2). cysteines [57] and putative Hypothetical proteins were 34.84% (n=14,657) holocyclotoxins [62] while pathogen-related proteins and transposable elements represent 0.23% (n=98) and 0.24% (n=106) respectively from all the SG_FA_CD contigs. Housekeeping products comprised of 8.69% (n=3,746) of the contigs. There were 1208 contigs (2.87%) encoding for secreted proteins. Most of the secreted proteins (26.82%) were enzymes.

Lipocalin 2.57% Pathogen 0.23% Protease Inhibitor Housekeeping 8.96% 17.88% Toxin like 4.06% Enzyme Novel 52.85% Immunity related 5.71% 26.90% Secreted 2.87% Antigen 1.08% Putative Secreted Hypothetical Glycine Rich 5.96% 23.51% 34.84% Mucin 1.32% Transposable Salivary 11% element 0.24%

Figure 2 – Functional classification of contig-encoded Ixodes holocyclus proteins in SG_FA_CD library. The overall contig proportion of different protein families/categories is shown, and all contigs categorized under “Secreted” were further divided into different subfamilies.

Lipocalin 11.47% Hypothetical 36.66% Glycine Rich 3.88% Enzyme Pathogen 19.71% 0.05% Mucin 2.26% Protease Inhibitor Secreted 10.99% 16.41% Novel 26.43% Salivary Toxin like 3.72% 22.29% Putative Immunity related 1.77% Secreted Housekeeping 23.26% Antigen 0.64% 20.44%

Figure 3 – Functional classification of contig-encoded Ixodes holocyclus proteins in SG_FA_BA library. The overall contig proportion of different protein families/categories is shown, and all contigs categorized under “Secreted” were further divided into different subfamilies.

B. 2. Functional annotations for Salivary Gland B.3 Contig representation in the two Ixodes collected from Female Adults collected from holocyclus next-generation libraries BAndicoots (SG_FA_BA). Comparison was made to study the Unlike contigs assembled from Illumina reads expression of different protein families from the generated from female adult ticks engorged on salivary gland of I. holocyclus collected from companion animals with paralysis symptoms, the different hosts: salivary glands collected from largest group of contigs in SG_FA_BA encoded for companion animals with paralysis symptoms hypothetical proteins, which is comprised of 36.66% (SG_FA_CD) and bandicoots (SG_FA_BA). In (n=1,383) of the all the contigs for the SG_FA_BA order to visualize the contig representation in each library (Figure 3). In the SG_FA_BA library, novel library and the comparisons, percentages of the six transcripts are the second most abundant group of protein family categories and secreted protein contigs and is made up of 26.43% (n=997) of the families were calculated to generate comparative total contigs, followed by housekeeping proteins graphs (Figure 4). (20.44%, n=771), secreted proteins (16.41%, n=619), and pathogen-related proteins (0.05%, n=2). Putative secreted proteins, which are predicted secreted proteins previously identified in other organisms, are the major groups of transcripts under secreted proteins (23.26%, n=144) in the SG_FA_BA library.

Comparison between SG_FD_CD and SG_FD_BA 60

50 SG_FD_CD (%) SG_FD_BA (%) 40

Contig representation (%) representation Contig 10

0 Housekeeping Pathogen Transposable Hypothetical Secreted Novel elements Category

Figure 4 – Comparing the abundance of six categories of protein families between salivary gland samples collected from adult female ticks engorged on paralysed companion animals (SG_FA_CD) and bandicoots (SG_FA_BA). Red bars represent the SG_FA_CD library while green bars represent the SG_FA_BA library.

Comparison of Secreted Protein Families between SG_FD_CD and SG_FD_BA 30.00

25.00 SG_FD_CD (%) SG_FD_BA (%) 20.00

15.00

10.00

5.00 Contig representation (%) representation Contig 0.00 Putative Salivary Mucin Glycine Enzyme Lipocalin Protease Toxin Antigen Immunity Secreted Rich Inhibitor like related

Secreted Protein Family

Figure 5 – Comparing the abundance of secreted protein families between salivary gland samples collected from adult female ticks engorged on paralysed companion animals (SG_FA_CD) and bandicoots (SG_FA_BA). Red bars represent the SG_FA_CD library while green bars represent the SG_FA_BA library.

B.4 Comparison of Salivary Gland collected from numbers of sequence reads and thus a reliable and Female Adults collected from paralysed Cats and robust transcriptome assembly for genome studies Dogs (SG_FA_CD) and Salivary Gland collected [48]. In this study, the sialome from I. holocyclus from Female Adults collected from BAndicoots was studied for the first time in the absence of a (SG_FA_BA) libraries reference genome. We employed both 454 and Illumina next-generation sequencing The SG_FA_CD and SG_FA_BA methodologies in this study. This is also the first libraries show significant differences in the paralysis tick sialome study, which provides a secreted and novel categories (Figure 4). For the preliminary resource and pioneering step towards a contigs representing secreted proteins, SG_FA_CD comprehensive understanding of paralysis tick yielded only 2.87%, which is approximately eight saliva composition. times less that SG_CD_BA, which yielded 16.41% of secreted proteins. In contrast, SG_FA_CD In order to counteract the host defense yielded 52.85% of contigs coding for novel system and achieve successful feeding, ticks have products, which is two times more than developed unique saliva compositions, which has SG_FA_BA, which yielded 26.43% novel proteins. been revealed in previous salivary gland Figure 5 further demonstrated the differences in the transcriptome (sialome) studies [49, 50]. The yields of different secreted protein families, transcriptome analysis conducted in this study significantly for salivary, enzyme, lipocalin, corroborated previous reports through the protease inhibitor and immunity-related secreted identification of ten secreted protein families proteins. (Table 5) reported in the two I. holocyclus cDNA IV. DISCUSSION libraries. These protein families were classified as:

Classical Sanger sequencing was utilized Antigen for most previous tick sialome studies [17, 24, 25, Two types of antigen families were 43-46] and only a few have used next-generation identified in I. holocyclus – antigen 5 and antigen sequencing [26, 47]. A comparison between P23. Members of antigen 5 protein families are I. ricinus transcriptomes generated using Sanger found in the venom of Vespid wasps and snakes and next-generation sequencing technologies and were identified previously in many discovered a higher and more comprehensive hematophagous tick transcriptomes [51, 52]. transcriptome coverage with next-generation However, most of the antigen 5 proteins have no sequencing methodologies, which is beneficial in known function. Antigen P23 is protein identified providing in-depth data towards understanding SG in I. scapularis and has demonstrated anti- transcript dynamics [17, 26]. The high coverage of coagulation activity [53]. next-generation transcriptome sequencing demonstrated a more accurate distribution of Enzymes sequence reads, which reflects the patterns of gene Some of the enzymes identified in the expression during tick feeding. In addition, even in saliva of hematophagous arthropods were shown to the absence of a reference genome, next generation assist with blood feeding. For example 5’ sequencing has been proven to produce sufficient nucleotidase, which is also called apyrase,

hydrolyses ATP and ADP released by damaged lipocalins are described to be involved in histamine cells to prevent activation of platelets and and serotonin binding [58], anti-haemostasis[59], neutrophils [15]. Metalloproteases are another large anti-complement activity [60], immunoglobulin group of hematophagy-assisting enzymes, which binding [61] and toxicity [62]. An extensive family inhibits the production of fibrin and fibrinogen [54]. of lipocalins is predicted to have more than 20 Endonuclease has been found in the saliva of the members and has a tendency of increasing in mosquito Culex pipiens quinquefasciatus. number with diverse functions being discovered in Endonucleases are predicted to have the ability to other ticks [62]. lower the viscosity of the skin matrix and allow diffusion of pharmacological components through Mucin the host dermis [22]. Chitinases may be involved in Mucins are putative glycoproteins with O- anti-fungal activity or housekeeping functions galactosylation sites and a chitin-binding domain associated with the cuticular structure [49]. Serine [63]. This group of proteins is predicted to serve as proteases may interrupt the fibrinolysis pathways in mechanical protection against pathogen invasion of the host or exert a prophenoloxidase anti-pathogen ticks, by coating the chitinous feeding mouthparts activity [49]. Other minor enzymes identified in the or the feeding lesion [49]. I. holocyclus adult female SGs include: inositol polyphosphate, phosphatase, carboxypeptidase, Protease Inhibitor sulfotransferase, dipeptidylpeptidase, leukotriene There are a few protease inhibitor protein hydrolase and phospholipase. families that can be characterized by their protease inhibitor domains such as the Kunitz domain, Glycine rich serpin domain and TIL (Trypsin Inhibitor Like) Glycine rich proteins have been identified domain. Kunitz domain containing protease in sialome studies of ticks and some were predicted inhibitors are one of the largest protein families in as salivary cement proteins that are utilized during tick saliva and they target proteases of the S1 tick-host attachment [49]. Another group of glycine family and enzymes complexes such as Xase and rich proteins are collagen-like proteins, which prothrombinase [49, 64]. Several members of function as part of the extracellular matrix of the Kunitz protease inhibitors are involved in anti- salivary glands and as part of the tracheolar system clotting [49], anti-thrombin [65] and anti-platelet [49]. GGY peptides, which are rich in glycine and [66] activities. Serpins control the clotting systems tyrosine, also belong to glycine rich protein family. of vertebrates by regulating serine protease GGY peptides are predicted to have an cascades. Serpins of I. ricinus, which are called antimicrobial function [55]. IRIS, are found to have immunosuppressive properties in cellular assays [67] and weak anti- Immunity related hemostatic function [68]. Another serpin, RMS-3, Immunity-related proteins are often found in R. microplus is predicted to counteract present in the saliva of hematophagous insects and immune responses during the host-parasite ticks [49]. There are two major groups of interaction [69]. The only characterized TIL immunity-related peptides - antimicrobial peptides domain containing protease inhibitor is ixodidin, and pattern recognition proteins. Antimicrobial which has antimicrobial, anti-trypsin and anti- peptides prevent contamination in the feeding elastase properties [70]. Cystatins (cysteine cavity and microbial growth in the ingested blood. proteinase inhibitors) are also a protease inhibitors, Examples of antimicrobial peptides are lysozyme, which mainly inhibit activities of peptidases of defensin, microplusin and histidine-rich peptides families C1 (papain family) and C13 (legumain [49]. Ficolin, ixoderin, peptidoglycan recognition family) [71]. Thyropins are another example of proteins and ML domain containing proteins are protease inhibitors. These members are found to examples of pattern recognition proteins. Ficolin contain Thyroglobulin type I repeats that may and ixoderins activate the invertebrate complement inactivate cysteine proteases. However, the system [56] while peptidoglycan recognition function of the thyropin family of inhibitors is still proteins are important for the activation of anti- unknown [49]. microbial enzymes [49]. ML domains enable the recognition of lipids important in innate immunity Putative secreted protein and lipid metabolism [49]. A significant number of contigs found in the I. holocyclus cDNA libraries matched Lipocalin previously described tick proteins, mainly also Lipocalins have a highly conserved barrel deducted from tick sialome studies. These putative structure, which enables them to bind and carry secreted proteins were found to have an open hydrophobic ligands. Lipocalins are ubiquitously reading frame in the previous studies but were not distributed and abundantly expressed in the functionally classified. The consistent presence of salivary glands of ticks [24, 25, 57]. In ticks, these groups of proteins in tick saliva suggests that

they have tick salivary gland specific functions yet holocyclus feeding on different host regulate gene to be determined. expression differently.

Salivary proteins The contig representation of the contig Salp15 is an example of salivary protein, counts utilized in this study would be an which was previously identified in I. scapularis. underestimation of the real read coverage. Further Salp15 was shown to inhibit CD4 T cells and to be analyses such as qPCR are needed to validate the essential for Lyme disease pathogen transmission abundances reported here. This transcriptomic [24]. Other salivary proteins are described as 8.9- study could be further improved by repeating the kDa proteins, 14-kDa proteins, 18.7-kDa proteins analysis of the samples with both 454 and Illumina and 22.5kDa proteins, which were previously technologies to provide replicate and more robust identified in other Ixodes ticks. These proteins have contig data. Currently, two samples were either an open reading frame and a signal peptide, processed using 454 or Illumina technologies. 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