Proteomic profiling of the midgut contents of Haemaphysalis flava

Ticks and Tick-borne Diseases xxx (xxxx) xxx–xxx

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Ticks and Tick-borne Diseases journal homepage: www.elsevier.com/locate/ttbdis

Proteomic profiling of the midgut contents of Haemaphysalis flava ⁎

Lei Liu, Tian-yin Cheng , Xiao-ming He College of Veterinary Medicine, Hunan Collaborative Innovation Center of Safety Production of Livestock and Poultry, Hunan Agricultural University, Changsha, China

A R T I C L E I N F O

A B S T R A C T

Keywords: Haemaphysalis flava Midgut contents Blood meal Digestion Proteome

Scant information is available regarding the proteins involved in blood meal processing in ticks. Here, we aimed to highlight the midgut proteins involved in preventing blood meal coagulation, and in facilitating intracellular digestion in the tick Haemaphysalis flava. Proteins were extracted from the midgut contents of fully engorged and partially engorged ticks. We used liquid chromatography tandem-mass spectrometry (LC–MS/MS) analysis to identify 131 unique peptides, and 102 proteins. Of these, 15 proteins, each with at least two unique peptides, were recognized with high confidence. We also retrieved 18 unigenes from our previous published transcriptomic libraries of the midguts and salivary glands of H. flava, and inferred the primary structures of nine proteins and fragments of five proteins. There were 23 and 21 unique proteins in the midgut contents of fully engorged and partially engorged ticks, respectively. We detected 58 shared proteins in the midgut contents of both fully engorged and partially engorged ticks. Of these, seven were significantly differentially expressed between fully engorged and partially engorged ticks: actin, calmodulin, elongation factor-1α, hsp90, multifunctional chaperone, tubulin α, and tubulin β. Our results demonstrated that the proteome of the midgut contents, combined with the transcriptome of the midgut, was a viable method for the reinforcement of protein identification. This method will facilitate further study of blood meal processing by ticks, as well as the identification of clues for tick infestation control. The existence of numerous proteins detected in the midgut contents also highlight the complexity of blood digestion in ticks; this area is in need of further investigation.

1. Introduction Haemaphysalis flava, a tick belonging to the family Ixodidae, has a broad geographic distribution in Asia. This species parasitizes human, cattle, wildlife, and a variety of birds. H. flava sheds both viral and bacterial pathogens, including thrombocytopenia syndrome, tick-borne encephalitis (Yun et al., 2016), Borrelia spp. (Ishiguro et al., 2000), Cercopithifilaria spp. (Nematoda: Onchocercidae) (Uni et al., 2013), Anaplasma, and Bartonella spp. (Kang et al., 2016). Previously, we characterized microbial community diversity in H. flava using NGS, and showed that eight bacteria were highly abundant throughout the life cycle of the tick (Rickettsia spp., Coxiella spp., Pseudomonas spp., Ehrlichia spp., Escherichia spp., Acinetobacter spp., Citrobacter spp., and Cupriavidus spp.; Duan and Cheng, 2017). As H. flava has a wide geographic distribution, and harbors dozens of pathogens through numerous hosts, this species is especially significant for public health. TickGARD and the Gavac vaccine, both based on the Bm86 antigen, are effective, economic, and environmentally friendly strategies for tick control (Rodríguez-Mallon, 2016). The identification of additional

antigen candidates for further vaccine development is critical. Highthroughput omics technologies, including proteomics and transcriptomics, have greatly facilitated this. To date, proteomic analyses of the saliva and feces of H. flava, as well as transcriptomic analyses of the midgut and salivary glands in this species, have identified several antigen candidates, including subolesin, hsc70, and enolase. As the host-tick-pathogen interface, the tick midgut is recognized as a reservoir of antigen candidates (Oleaga et al., 2017); Bm86 is expressed there. Midguts are also major sites for the storage and digestion of blood meals in ticks. Although the molecular physiology of blood meal processing in the midgut epithelia has received intense study (Sojka et al., 2013), the molecules and mechanisms in the midgut that prevent blood meal coagulation and that mediate intracellular digestion remain largely unknown. As blood-proteins provide ticks with essential nutrients, gut contents, which may include food residues, molecules released by upper tissues, and microorganisms, are directly associated with digestion and the efficient utilization of blood meals; gut contents are thus closely related to tick vitality and vector capability (Connat, 1991). However, integrated analyses of tick midgut contents are

Abbreviations: ACN, acetonitrile; BCA, bicinchoninic acid; DTT, DL Dithiothreitol; FASP, filter aided sample preparation; FDR, false discovery rate; HCD, higher-energy collisional dissociation; KO, knocked out; LC–MS/MS, liquid chromatography tandem-mass spectrometry; LFQ, label free quantification; NGS, next generation sequencing; SDS, sodium dodecyl sulfate; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; UA buffer, urea acid buffer; Vitellogenin, Vg ⁎ Corresponding author at: College of Veterinary Medicine, Hunan Agricultural University, Changsha, 410128, China. E-mail address: [email protected] (T.-y. Cheng). https://doi.org/10.1016/j.ttbdis.2018.01.008 Received 20 August 2017; Received in revised form 6 January 2018; Accepted 13 January 2018 1877-959X/ © 2018 Published by Elsevier GmbH.

Please cite this article as: Liu, L., Ticks and Tick-borne Diseases (2018), https://doi.org/10.1016/j.ttbdis.2018.01.008

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analytical column (EASY column SC200 150 μm × 100 mm (RP-C18)). The flow rate was set to 250 nL/min. Mobile phase B was used as the eluent. After HPLC separation, both samples were analyzed with a QExactive mass spectrometer (Thermo Scientific, MA, USA). Nano spray ionization was used as the ion source, and argon was used as the collision gas. We set the analysis time to 120 min. MS data was obtained using a data-dependent top10 method, where we dynamically chose the most abundant precursor ions from the survey scan (300–1 800 m/z) for higher-energy collisional dissociation (HCD) fragmentation. Determination of the target value was based on predictive automatic gain control. Dynamic exclusion duration was 25 s. Survey scans were acquired at a resolution of 70 000 at m/z 200, and the resolution for the HCD spectra was set to 17 500 at m/z 200. Normalized collision energy was 30 eV, and the underfill ratio was defined as 0.1%. The mass spectrometer was run with peptide recognition mode enabled. We performed three biological and three technical replicates of the LC–MS/MS analysis.

extremely rare. We therefore aim to identify the proteins in the midgut of H. flava with LC–MS/MS, and to combine these identifications with our previous published transcriptome data from the midgut and salivary glands of this species. Using these two datasets, we aim to determine the primary structures of related proteins. These structures will provide a basis for further study of the mechanisms of blood meal anticoagulation and digestion, as well as a framework for the identification of feasible antigen candidates for future vaccine development. 2. Materials and methods 2.1. Tick specimens Live H. flava specimens were collected in Xinyang, Henan Province, China (32°13′N,114°08′E). Identification was confirmed using morphological and molecular analysis (Yan and Cheng, 2015). Ticks were fed on hedgehogs in our laboratory at Hunan Agricultural University (HUNAU), Hunan Province, China. All experimental procedures were approved and overseen by Institutional Animal Care and Use Committee at HUNAU (No.43321503). No animals were subject to unnecessary suffering in the present study.

2.4. MS data analysis MS data were processed with MaxQuant (version 1.3.0.5). We identified proteins by searching against the Uniprot database uniprot_Ixodidae_80119_20151102. We constructed fasta and putative peptide libraries based on our previously published transcriptomes of the midgut and salivary glands of H. flava. Carbamidomethylation of cysteine was used as a fixed modification, while oxidation of methionine was defined as a variable modification. Searches were carried out with tryptic specificity, allowing two missed cleavage sites at most, and a mass measurement tolerance of 20 ppm in MS mode and 0.5 Da for MS/MS ions. The global false discovery rate (FDR) cutoff for peptide and protein identification was set to 0.01. Hits with at least two unique peptides were considered proteins with high confidence. Label-free quantification (LFQ) was carried out in MaxQuant as previously described by Luber et al. (2010). Protein abundance was calculated on the basis of the normalized spectral protein (LFQ) intensity. For each protein common to the two engorgement states, we calculated the LFQ intensity ratio between fully engorged and partially engorged. We also compared LFQ intensities using the Student’s t test. The difference between LFQ intensities was considered significant if the LFQ ratio was ≥ 1.5 or ≤0.67, and p was < 0.05. The raw data files used for these analyses are available from the integrated proteome resource (http://iprox.org/page/SSV024. html;url=1511751279689Dh3L with the key: zORj; project ID: IPX0001041003).

2.2. Collection and pretreatment of midgut contents We dissected 12 adult female ticks, six fully engorged and six partially engorged, under a stereo microscope. Midgut contents, about 30 μL from each tick, were carefully squeezed into a clean tube containing 90 μL sodium citrate-physiological saline. Tubes were vortexed and centrifuged at 5 000 rpm for 15 min. We removed 4 μL aliquots of each supernatant, and pooled these in two groups based on the feeding state of the source tick: fully engorged or partially engorged. We added an equal volume of SDT buffer (4% SDS, 100 mM DTT, and 150 mM Tris-HCl pH 8.0) to each supernatant pool. We vortexed the supernauts, and placed the tubes in a water bath at 100 °C for 5 min. We preliminarily analyzed the proteins in the two pools with the conventional BCA method and with SDS-PAGE (Song et al., 2016). 2.3. Protein analysis with LC–MS/MS A filter aided sample preparation (FASP) was used to purify and digest proteins before instrumental analysis (Wiśniewski et al., 2009). Briefly, 200 μL UA buffer (150 mM Tris-HCl and 8 M urea pH 8.0) was added to 10 μL each midgut extract (not pooled), and well vortexed. Samples were then transferred to ultrafiltration tubes fitted with 10 kDa membranes, and centrifuged at 14,000×g for 15 min. Proteins remained on the membrane, and the filtrate was discarded. We performed this filtration twice to maximize the removal of impurities. Residues on the membrane were reconstituted in 200 μL UA buffer containing 50 mM iodoacetamide, shocked at 600 rpm for 1 min, and allowed to stand for 30 min at room temperature in the dark. Samples were centrifuged at 14 000g for 10 min. Filtrate was discarded, and residue retained on the membrane was washed twice with 200 μL UA buffer, and then with 200 μL dissolution buffer (25 mM NH4HCO3). Protein resides were digested with 40 μL trypsin buffer (3 μg trypsin (Promega, WI, USA) in 40 μL 25 mM NH4HCO3) in a 37 °C water bath for 16–18 h. This digest was transferred to clean ultrafiltration tubes fitted with 10 kDa membranes, and centrifuged at 14,000×g for 10 min. We collected 5 μL filtrate from each sample, and measured the peptide levels at OD280. We diluted each filtrate sample to 1 μg/μL peptide, and 5 μL of each diluted sample was loaded onto an EASY-nLC1000 system (Thermo Scientific, MA, USA). Mobile phase A was 0.1% formic acid, and mobile phase B was 84% acetonitrile in 0.1% formic acid. Chromatographic columns were balanced with 95% mobile phase A before sample loading. Samples were injected onto a trap column (Thermo EASY column SC001 traps 150 μm × 20 mm (RP-C18)), and then onto an

2.5. Bioinformatics analysis of identified proteins We used our protein identifications and annotations to retrieve unigenes from the previously published H. flava transcriptome libraries of the midgut (NCBI Gene Expression Omnibus ID: GSE69721; https:// www.ncbi.nlm.nih.gov/gds/?term=GSE69721) and the salivary glands (NCBI Gene Expression Omnibus ID: GSE67247; https://www.ncbi.nlm. nih.gov/gds/?term=GSE67247). The primary structures of the proteins were predicted based on gene sequences. 3. Results 3.1. Protein analysis with SDS-PAGE Our BCA indicated that there was 9.8 μg/μL protein in the midguts of partially engorged ticks, and 22.4 μg/μL protein in the midguts of fully engorged ticks. SDS-PAGE protein bands were clear, and ranged from 10 to 170 kDa (Fig. 1). Protein band patterns were generally similar between engorgement states: both states had very intense bands at about 70 kDa (Fig. 1). Our analyses therefore indicated that our midgut protein extraction was effective. 2

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previously published midgut and salivary glands transcriptomic datasets (GSE69721 and GSE67247; Table 2). The proteins encoded by these unigenes were predicted with DNAman (version 9). It is noteworthy that Contig39 encoded calmodulin; Contig390 encoded elongation factor-1α; Contig1683 encoded GAPDH; Contig352 encoded GTPbinding nuclear protein; Contig580 encoded histone H4; Contig1176 encoded hsp70; ContigF166 encoded multifunctional chaperone; Contig5055 encoded ubiquitin; and Contig2799 encoded tubulin β. These results indicated that these proteins might be intentionally secreted by the tick midgut. All protein structures were highly conserved, with the exception of Vg-1 and Vg-2. 3.4. Comparison of protein quantity between fully engorged and partially engorged ticks Of the 102 proteins we identified, 58 were detected in the midgut contents of both fully engorged and partially engorged ticks. There were 23 and 21 unique proteins in midgut contents of fully engorged ticks and partially engorged ticks, respectively. Based on LFQ intensity, the relative abundances of seven of the 58 common proteins were significantly different between fully engorged and partially engorged ticks (LFQ ratio > 1.5 or < 0.67; p < 0.05). Four of these eight were proteins with high confidence: GTP-binding nuclear protein, hsp90, multifunctional chaperone and tubulin α (Table 3). 4. Discussion

Fig. 1. Proteins extracted from the midgut contents of Haemaphysalis flava. M, markers; Lane 1, proteins extracted from the midgut contents of fully engorged ticks; Lane 2, proteins extracted from the midgut contents of partially engorged ticks.

Ticks feed on host blood; these blood meals provide nutrition and energy for growth and development (Anderson and Magnarelli, 2008). Blood meals remain in the tick midgut for extended periods of time in those species with a long life cycle. During this time, complicated biochemical reactions prevent blood clotting and facilitate somatic cell uptake for intracellular digestion (Assumpção et al., 2016; Oleaga et al., 2017). However, the molecular mechanisms supporting these processes have not been fully described. Here, we differentiated and identified 15 proteins with high confidence in the midgut contents of H. flava using LC–MS/MS. We combined these protein identifications with our previously published midgut and salivary gland transcriptome datasets to identify unigenes and to predict primary protein structures. However, it is noteworthy that we just have used the ixodidae database to identify tick proteins, but have not searched the erinaceinae database to identify host proteins because of its unavailability. The high confidence proteins that we identified were implicated in five types of functions: formation of

3.2. LC–MS/MS analysis We detected 131 unique peptides with LC–MS/MS, ranging in length from 7 aa to 51 aa. We identified 102 proteins based on these unique peptides; 15 of these had at least 2 unique peptides, and were thus recognized with high confidence (Table 1): actin, valosin containing protein, calmodulin, elongation factor-1α, GAPDH, GTPbinding nuclear protein, histone H4, hsp70, hsp90, multifunctional chaperone, ubiquitin, vitellogenin-1 (Vg-1), Vg-2, tubulin α, and tubulin β.

3.3. Identification of corresponding genes We identified 18 unigenes based on our protein annotations and our

Table 1 Proteins identified with high confidence from the midgut content proteome of Haemaphysalis flava. No.

Unique peptide

Peptides deduced from

Coverage (%)

1

AVFPSIVGR, DLYANTVLSGGTTMYPGIADR, DSYVGDEAQSK, LCYVALDFEQEMATAASSSSLEK, SYELPDGQVITIGNER, VAPEEHPVLLTEAPLNPK LAGESESNLR LDQLIYIPLPDEK EAFSLFDKDGDGTITTK, ADQLTEEQIAEFK EHALLAYTLGVK, IGGIGTVPVGR IVSNASCTTNCLAPLAK, GAAQNIIPASTGAAK, VPTPNVSVVDLTCR CVLVGDGGTGK, VCENIPIVLCGNK DNIQGITKPAIR, TVTAMDVVYALK ARFEELNADLFR, DAGTIAGLNVLR, IINEPTAAAIAYGLDKK, STAGDTHLGGEDFDNR ADLINNLGTIAK, GVVDSEDLPLNISR AAFDDAIAELDTLSEESYK, EAAENSLVAYK, NLLSVAYK IQDKEGIPPDQQR, TITLEVEPSDTIENVK, ESTLHLVLR EPTEILFDEFYR, SEYPCHFELSQHLR LLNGLVGPQPGSTK LCGLCGDYNLDR NLDIERPTYTNLNR, VGINYQPPTVVPGGDLAK QLFHPEQLITGK ALTVPELTQQMFDAK, GHYTEGAELVDSVLDVVR

cds. Contig240 in GSE69721

27.3

cds. Contig2872 in GSE69721 cds. Contig16282 in GSE69721 cds. Contig39 in GSE69721 cds. Contig390 in GSE69721 cds. Contig1683 in GSE69721 cds. Contig352 in GSE69721 cds. Contig580 in GSE69721 cds. Contig1176 in GSE69721 cds. Contig1572 in GSE69721 cds. Contig F166 in GSE69721 cds. Contig5055 in GSE69721 cds. Contig12613 in GSE67247 cds. Contig10258 in GSE69721 cds. Contig1122 in GSE67247 cds. Contig34 in GSE69721 cds. Contig514 in GSE69721 cds. Contig2799 in GSE69721

2.2 5.3 16.7 4.8 13.0 10.0 22.4 8.9 6.7 14.7 18.7 1.7 5.5 4.3 10.4 7.3 7.0

2 3 4 5 6 7 8 9 10 11 12 13 14 15

3

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Y Y Y N N Y

Y Y Y Y Y Y Y Y Y Y Y Y N N

3 037 1 824 1 293 849 5 659 1 159 1 491 2 238 875 2 340

1197 1476 842 1513 1899 1178 1030 799 2 163 1164 2719 760 4694 1064 932 1359 492 1417

359 333 224 149 484 353 239 103 627 388 256 171 1564 256 310 320 164 445

Actin, Amblyomma cajennense, A0A023FIL9 Valosin containing protein, Haemaphysalis longicornis, A7BFI9 Valosin containing protein, H. longicornis, A7BFI9 Calmodulin, Rhipicephalus appendiculatus, A0A131Z1F6 Elongation factor-1α, A. aureolatum, A0A1E1XAX2 GAPDH, A. triste, A0A023GLP1 GTP-binding nuclear protein, Ornithodoros moubata, A0A1Z5KX72 Histone H4, A. americanum, A0A0C9QYX1 Hsp70, R. appendiculatus, A0A131YWH5 Hsp90, A. cajennense, A0A023FL50 Multifunctional chaperone, Hyalomma excavatum, A0A131XIE5 Ubiquitin, A. variegatum, F0J9K1 Vitellogenin-1, H. longicornis, E1CAX9 Vitellogenin-2, H. longicornis, B1B544 Vitellogenin-2, H. longicornis, B1B544 Tubulin α, R. appendiculatus, A0A131YP40 Tubulin α, R. appendiculatus, A0A131YP40 Tubulin β, R. appendiculatus, A0A131YRB1

0 0 5.5e–163 1.5e–99 0 0 1.9e–160 4.6e–67 0 0 7.60e–178 2.7e–112 0 1.40e–143 0 0 2.90e–113 0

1 811 1 780 1 245 758 2 345 1 603 1 173

99.7 89.6 99.2 100 98.9 91.3 98.2. 100 93.1 88.9 100 97.7 86.2 84 86 99.1 99.4 99.1

N N N Y Y Y Y Y N N Y Y N N

cellular structure (actin, tubulin α, and tubulin β), cellular signal transduction (GTP-binding nuclear protein, calmodulin, and elongation factor-1), substance metabolism (GAPDH, ubiquitin, and valosin containing protein), molecular chaperoning (hsp70, hsp90, and multifunctional chaperone), and egg formation (Vg-1 and Vg-2). Studies have identified the structural and functional roles of actin, elongation factor-1α, ubiquitin, valosin-containing protein, Vg-1, and Vg-2 in ticks. Actin, a 376 aa protein that is a constituent of myofibril, is conserved in the ticks Ornithodoros moubata, Rhipicephalus microplus, H. longicornis, and R. appendiculatus (Da et al., 2005; Horigane et al., 2007). Actins may be of subtype α, β or γ; our analysis of Contig240 from midgut transcriptomic library indicated that the actins in the H. flava midgut contents were β-actin. Our quantitative protein analysis indicated that β-actin levels in the midgut contents were not statistically different between fully engorged ticks and partially engorged ticks. This result is consistent with a previous study, where the expression of β-actin, elongation factor 1α, and GAPDH in the ticks R. microplus and R. appendiculatus was stable across various life stages (Nijhof, 2009). We found that the product of Contig390 was 462 aa in length, and was more than 97% identical to elongation factor-1α in R. microplus, Amblyomma cajennense, and I. scapularis, indicating that this protein is highly conserved in ticks. As expression of elongation factor-1α is stable across life stages (Nijhof, 2009), it may be a house-keeping gene. de la Fuente et al. (2008) cloned the G II and G III zones of elongation factor1α in I. scapularis, and showed that the G II zone was involved in subolesin-mediated regulation of gene expression. Inhibiting the G II zone with RNAi lead to significant reductions in engorgement, longevity, and egg viability (Almazán et al., 2010; de la Fuente et al., 2008). The products of Contig5055 were highly conserved, polymerized ubiquitin monomers. Crampton et al. (1998) inhibited ubiquitin in R. microplus and R. annulatus with dsRNA viruses derived from ubiquitin, again resulting in reduced engorgement, longevity, and egg viability. However, peptide fragments derived from ubiquitin did not work effectively (Almazán et al., 2010). Valosin containing protein, a member of the AAA+ class family, is a multifunction molecular chaperone with Walker A (GX4GKT) and Walker B (Hy4DEX2) motifs (Ogura and Wilkinson, 2001). Based on the structure of the products of Contig2872 and Contig16282, valosin containing protein in H. flava possessed these two signatures. In H. longicornis, valosin containing protein binds specifically to antiserums; RNAi resulted in a 42% reduction in full engorged weight (Boldbaatar et al., 2007). Vitellogenin is a protein restricted to female ticks; in H. longicornis, vitellogenin is found as Vg-1, Vg-2 and Vg-3. Vg-1 is expressed mainly in the midgut, while Vg-2 and Vg-3 are expressed in the fat bodies. Vg-2 is also expressed in the ovaries. RNAi trials suggested that interference with any of the three vitellogenins caused reductions in engorgement, longevity, and spawning rate in H. longicornis (Boldbaatar et al., 2010); similar effects were observed in A. hebraeum (Smith and Kaufman, 2014). We detected genes expressing both Vg-1 and Vg-2 in the midgut transcriptome of H. flava, indicating that the Vg-1 and Vg-2 expressed in the midgut were not secreted into hemolymph once expressed. The gene sequences of Vg-1 and Vg-2 were complicated and variable, making these potential candidate antigens for vaccine development. Although the functional roles of histone H4 and hsp70 in ticks are unknown, studies in other species suggest they may be involved in preventing blood meal coagulation. Histone H4 was highly conserved, with 100% identity between H. flava and a variety of species including humans, rodents, cattle, goats, chickens, and flies. Histone H4 increases the formation of thrombin by increasing the efficiency of prothrombin activation by FXa, leading to faster blood clotting (Barranco-Medina et al., 2013; Pozzi and Cera, 2016). Hsp70, a member of the heat-shock protein family (Kiang and Tsokos, 1998), might impact blood coagulation through the regulation of platelet degranulation, aggregation, and integrin activity (Rigg et al., 2016). HSPA1A/B KO mice were prone to thrombus formation (Allende

15

14

3 4 5 6 7 8 9 10 11 12 13

cds. cds. cds. cds. cds. cds. cds. cds. cds. cds. cds. cds. cds. cds. cds. cds. cds. cds. 1 2

Contig240 in GSE69721 Contig2872 in GSE69721 Contig16282 in GSE69721 Contig39 in GSE69721 Contig390 in GSE69721 Contig1683 in GSE69721 Contig352 in GSE69721 Contig580 in GSE69721 Contig1176 in GSE69721 Contig1572 in GSE69721 Contig F166 in GSE69721 Contig5055 in GSE69721 Contig12613 in GSE67247 Contig10258 in GSE69721 Contig1122 in GSE67247 Contig34 in GSE69721 Contig514 in GSE69721 Contig2799 in GSE69721

Similar peptide fragment Peptide length (aa) Unigene length (bp) Contig from No.

Table 2 Unigenes encoding high-confidence proteins from the midgut contents proteome of Haemaphysalis flava.

E-value

Score

Identity (%)

Integrity (Y/N)

Conserved (Y/N)

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Table 3 Quantitative analysis of proteins with high confidence in the midgut contents of fully engorged and partially engorged female Haemaphysalis flava. Protein

Actin Valosin containing protein Calmodulin Elongation factor-1α GAPDH GTP-binding nuclear protein Hsp70 Hsp90 Histone H4 Multifunctional chaperone Ubiquitin Vitellogenin-1 Vitellogenin-2 Tubulin α Tubulin β

LFQ intensity (x ± SD)

Full engorged vs Partially engorged

Partially engorged

Fully engorged

Ratio

T-test

324.37 ± 26.28 10.89 ± 1.28 49.41 ± 4.84 13.06 ± 2.39 112.77 ± 45.09 9.84 ± 3.73 52.69 ± 6.00 18.15 ± 3.46 9.37 ± 4.38 10.12 ± 0.97 279.28 ± 19.19 3.26 ± 0.54 22.94 ± 3.81 10.27 ± 1.48 0.81 ± 0.06

392.37 ± 18.73 11.31 ± 1.28 69.04 ± 8.66 8.76 ± 2.04 117.22 ± 23.79 13.26 ± 2.42 57.99 ± 4.89 12.16 ± 2.77 1.95 ± 3.36 0.00 ± 0.00 274.98 ± 21.25 3.13 ± 0.15 21.94 ± 3.65 39.42 ± 8.83 0.96 ± 0.09

1.21 1.04 1.39 0.67 1.04 1.35 1.10 0.67 0.21 +∞ 0.98 0.96 0.96 3.84 1.18

p < 0.01 p = 0.58 p < 0.01 p < 0.01 p = 0.66 p = 0.09 p = 0.12 p < 0.01 p = 0.73 p < 0.01 p = 0.72 p = 0.59 p = 0.65 p < 0.01 p = 0.01

Note

downregulated

downregulated downregulated

upregulated

Acknowledgment

et al., 2016). Triatomine bugs with hsc70 (a member of the hsp70 family) KO had reduced engorgement and high mortality (Paim et al., 2016). However, it has been suggested that hsp70 might be unsuitable as a protective antigen (Tian et al., 2011). Indeed, in H. longicornis, hsp70 encoded a 661 aa protein with a KDEL motif at the C terminal: an ER-type hsp70 (Tian et al., 2011). It was shown that this type of recombinant hsp70 did not had a protective effect on vaccinated rabbits (Tian et al., 2011). We discovered that the full-length of hsc70 in H. flava was 648 aa, and its expression was high in salivary glands and midguts of partially engorged ticks (Liu et al., 2017). Previously, we cloned the cDNA of hsp90 based on H. flava Contig1572. This gene was 2 475 bp and encoded a 738 aa protein (GenBank accession no.: MF66449.1). Here, we identified family signatures like NKEIFLRELISNSSDALDKIR, LGTIAKSGT, GQFGVGFYSAYLVAD, IKLYVRRVFI and GVVDSEDLPLNISREM in the primary structure of hsp70, as well as a conserved MEEVD motif at the C-terminus. The functional roles of heat-shock proteins in ticks need further investigation. Comparison of blast results among H. flava and monkeys, pigs, cattle, goats, mice, and chickens indicated that histone H4 and ubiquitin were highly conserved. Therefore, these two are not candidate antigens. Other proteins (including α-tubulin, β-tubulin, GTP-binding nuclear protein, calmodulin, elongation factor-1α, valosin containing protein, hsp90, hsp70, and multifunctional chaperone) were also well conserved among animal species, despite some variable regions. It was difficult to determine, based on the peptides detected with MS, whether a given protein originated in the tick itself or in the host. Consequently, it was also difficult to quantify expression differences between proteins at different feeding states. It is noteworthy that GAPDH is a versatile molecule typically involved in monosaccharide glycolysis, energy generation, and iron transport. In ticks, the role of GAPDH is not yet characterized (Kumar et al., 2012). It might be that GAPDH is a potential antigen against Onchocerca volvulus and Schistosoma japonicum (Erttmann et al., 2005; Waine et al., 1993). Our results demonstrated that the midgut content proteome, combined with the midgut transcriptome, is a viable method with which to reinforce protein identification. The numerous proteins detected in the midgut contents also highlight the complexity of the blood digestion process in ticks; more study of this process is sorely needed.

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Funding The work was supported by the National Natural Science Foundation of China (No. 31372431) and the State Key Laboratory of Veterinary Etiological Biology (No. SKLVEB2016KFKT009). 5

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