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Article

Egg Protein Compositions over Embryonic Development in Haemaphysalis hystricis Ticks

by
Qiwu Tang
,
Tianyin Cheng
* and
Wei Liu
*
College of Veterinary Medicine, Hunan Agricultural University, Changsha 410128, China
*
Authors to whom correspondence should be addressed.
Animals 2024, 14(23), 3466; https://doi.org/10.3390/ani14233466
Submission received: 28 October 2024 / Revised: 27 November 2024 / Accepted: 29 November 2024 / Published: 30 November 2024
(This article belongs to the Special Issue Parasite Epidemiology and Molecules Identification in Wild and Domestic Animals)
Browse Figures
Figure 1
<p>SDS-PAGE analysis of protein extract from <span class="html-italic">H. hystricis</span> eggs. A total of 80 μg of protein extract was loaded per sample. Lane M represents the 15–250 kDa molecular weight marker. D1, D7, D14, and D21 correspond to eggs incubated for 1, 7, 14, and 21 days, respectively. Bands 1–12 indicate the protein bands excised for further analysis.</p> "> Figure 2
<p>Top 4 most prominent proteins identified in 12 bands from day-one eggs.</p> "> Figure 3
<p>Analysis of protein dynamics in <span class="html-italic">H. hystricis</span> tick eggs across different incubation days. Each sample was spiked with the stable isotope iRT KIT peptide as an internal standard. Tryptic peptides were analyzed using the nLC-1200 system. Protein abundances at 7, 14, and 21 days of incubation were normalized to the levels observed at day. D1, D7, D14, and D21 correspond to eggs incubated for 1, 7, 14, and 21 days, respectively.</p> ">
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Simple Summary
Ticks, as obligate ectoparasites, pose significant health risks by transmitting pathogens to various hosts, including humans and livestock, resulting in diseases and economic losses. This study focuses on Haemaphysalis hystricis, a tick prevalent in Southeast Asia, which infests both wild and domestic animals, leading to severe health impacts like emaciation and growth stunting. The tick’s life cycle, which includes stages from egg to adult, is crucial for understanding its development and for devising effective control strategies. This research aims to characterize protein expression in H. hystricis eggs throughout embryonic development using LC/MS/MS techniques. By identifying key proteins involved in egg development and metabolism, the study seeks to provide insights into tick embryogenesis and to identify potential targets for interventions that could disrupt tick reproduction and reduce the transmission of tick-borne diseases. Ultimately, the findings are expected to contribute to improving public health and animal husbandry practices by informing better tick management strategies.
Abstract
Tick eggs contain a series of proteins that play important roles in egg development. A thorough characterization of egg protein expression throughout development is essential for understanding tick embryogenesis and for screening candidate molecules to develop novel interventions. In this study, eggs at four developmental stages (0, 7, 14, and 21 incubation days) were collected, and their protein extraction was profiled using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). On the first day of egg protein extraction, protein bands from day-1 eggs were re-collected and subsequently analyzed using liquid chromatography–tandem mass spectrometry (LC-MS/MS). The dynamic changes in forty egg proteins during development were further investigated using LC-parallel reaction monitoring (PRM)/MS analysis. A total of 108 transcripts were detected in day-1 eggs. Based on protein functions and families, these transcripts were classified into eight categories: transporters, enzymes, immunity and antimicrobial proteins, proteinase inhibitors, cytoskeletal proteins, heat shock proteins, secreted proteins, and uncharacterized proteins. Identification of the protein bands revealed that nine bands predominantly consisted of vitellogenin and vitellin-A, while other notable proteins included cathepsins and Kunitz domain-containing proteins. LC-PRM/MS analysis indicated that 28 transcripts increased significantly in abundance, including 13/18 enzymes, 1/1 antimicrobial peptide, 2/2 neutrophil elastase inhibitors, 3/4 vitellogenins, 3/3 heat shock proteins, 3/3 cytoskeletal proteins, 1/1 elongation factor-1, and 1/1 uncharacterized protein. Conversely, five transcripts showed a decrease significantly, including 1/1 Kunitz domain-containing protein, 2/6 aspartic proteases, and 2/5 serpins. This research provides a comprehensive overview of egg proteins and highlights the dynamic changes in protein expression during embryonic development, which may be pivotal for understanding protein functions and selecting potential candidates for further study.

1. Introduction

Ticks, obligate ectoparasites, infest a wide range of hosts, including mammals, birds, and reptiles, such as wild deer, rodents, and various bird species, along with domestic animals like dogs and livestock. Renowned for transmitting pathogenic microorganisms—such as protozoa (e.g., Babesia spp.), bacteria (e.g., Borrelia, Rickettsia, Ehrlichia spp.), and viruses—they cause significant health issues, including anemia, dermatitis, and the transmission of tick-borne diseases, which affect both human and animal health, leading to economic losses in livestock production. Effective tick population management and disease prevention are therefore essential to mitigating their impact on public health and animal husbandry.
Haemaphysalis hystricis [1], an Ixodidae family member, is widespread in the temperate and subtropical hilly and mountainous regions of Southeast Asia, including Japan [2], Malaysia [3], and Indonesia [4], and has also been recorded in several Chinese provinces such as Taiwan [5], Wuhan [6], and Sichuan [7]. This tick species predominantly parasitizes wild hosts like wild boars [3,8], hedgehogs, pangolins [9], and giant pandas [10], as well as domestic animals, including cattle, goats [6], and dogs [2,7]. Infestation can result in host emaciation and stunted growth, while ticks act as vectors for various pathogens, including Trypanosoma [11], Ehrlichia, Rickettsia [6,12], Borrelia [3], and Babesia [5,13].
The tick life cycle encompasses four stages: egg, larva, nymph, and adult. During the egg stage, fertilization initiates complex developmental processes—cleavage, blastulation, gastrulation, and segmentation—culminating in larval emergence. These intricately regulated stages are key to understanding embryogenesis and devising advanced tick control strategies.
A thorough characterization of egg protein expression throughout development is essential for elucidating tick developmental mechanisms. Previous studies have characterized protein profiles of tick salivary glands and saliva, midgut, midgut contents, and eggs [14,15,16,17,18]. Several proteins involved in egg development across different tick species have been identified, including nutrient-related proteins like yolk protein and vitellogenin (also functioning as a nutrient in eggs) and lipocalin; enzymes such as heme-binding aspartic proteinases; vitellin-degrading cysteine endopeptidases; cathepsin L-like proteinases [19,20,21]; protease inhibitors like serpin, cysteine, and Kunitz domain-containing proteins; and immune proteins including antimicrobial peptides (AMP), macroglobulin, and cysteine-rich proteins. Additionally, enzymes such as GST, which exhibit free radical scavenging activity [22]; serine protease inhibitors [23]; and antimicrobial peptides [24] have been identified. Investigating the egg development of H. hystricis can enhance the understanding of tick embryogenesis and aid in screening candidate molecules for developing novel interventions that disrupt tick reproduction and pathogen transmission. This study employs LC/MS/MS methods to identify protein components in the protoplasm of H. hystricis eggs and analyze the dynamic changes in protein expression during egg development.

2. Materials and Methods

2.1. Collection of Ticks and Eggs

Xiangxi County, located in western Hunan Province, China, spans latitudes 27°4′ N to 29°5′ N and longitudes 108°6′ E to 110°6′ E, and is predominantly mountainous and hilly. Ticks were collected from wild boars (Arctonyx collaris) that had been rescued by the local wildlife protection department and kept at a wild animal farm in Xiangxi. The sampling was conducted from July to October 2022, with ticks being carefully removed using tweezers. The collection process was approved by the Hunan Provincial Department of Animal Protection and supervised by the Hunan Animal Health and Usage Committee (No. 43321503), ensuring no harm to the animals.
Ticks were gathered by local residents from the wild boars and transported to the laboratory in ventilated plastic bottles. Identification was performed based on morphological characteristics [25] observed under a light microscope, with confirmation via molecular methods following the protocol by Ernieenor et al. [26]. Concurrently, the ticks were weighed, and 20 engorged H. hystrici females, each weighing approximately 350 mg, were selected. These ticks were placed individually in wells of a 6-well plate, which was then maintained in complete darkness at 28 °C and 85% relative humidity within an incubator.
Eggs laid by the ticks were collected and weighed daily at a consistent time until the ticks died. The collected eggs were pooled and divided into four aliquots of roughly 50 mg each. One aliquot underwent a dewaxing process using a 1.5 mL chloroform/methanol mixture (2:1) for 15–20 s [27], followed by vortexing and careful removal of the supernatant. Afterward, 1 mL of sterile deionized water was added, the sample was vortexed again for 15–20 s, and the supernatant was discarded. The eggs were then labeled with the date and promptly stored in liquid nitrogen.
The remaining three aliquots were incubated under identical conditions of darkness, temperature (28 °C), and humidity (85% RH). Eggs were collected on days 7, 14, and 21 of incubation, dewaxed, and stored in liquid nitrogen. However, the incubation period may vary depending on climate conditions in the field [28,29].

2.2. Egg Protein Extraction

For further processing, dewaxed eggs from days 0, 7, 14, and 21 were transferred to sterilized glass homogenizers. Each sample was combined with 0.3 mL of sterile saline and ground thoroughly. The homogenate was then transferred to 1.0 mL centrifuge tubes, with an additional 0.5 mL of sterile saline added. Samples were kept at 4 °C for 30 min, followed by centrifugation at 5000 rpm for 5 min. The supernatant was carefully collected for subsequent analyses.
Supernatants were mixed with lysis buffer containing 4% SDS, 0.1 M DTT, 150 mM Tris-HCl (pH 8.0), and 0.1 mL of a protease inhibitor cocktail (P9599, Sigma, St. Louis, MI, USA). The mixture was boiled for 3 min and sonicated on ice. The resulting crude extract was reheated in boiling water and centrifuged at 16,000× g for 10 min at 4 °C to clarify the solution. Protein concentration was measured using the BCA protein assay reagent (Bio-Rad, Hercules, CA, USA). Finally, the supernatants were stored at −80 °C for future use.

2.3. SDS-PAGE from Four Stages Egg

A 20 μg aliquot of egg protein supernatant from each sample was combined with 30 μL of SDT buffer (4% sodium dodecyl sulfate, 100 mM dithiothreitol, and 150 mM Tris-HCl, pH 8.0). The mixture was subjected to ultrasonication under the following conditions: 80 watts for 10 s, followed by a 15 s rest, repeated 10 times. Post-ultrasonication, the mixture was boiled for 5 min. After cooling to room temperature, it was centrifuged at 14,000× g for 10 min at 4 °C. SDS-PAGE analysis was conducted with 5% stacking and 10% separating gels, where the supernatant from the first day and 10 μL of boar blood serum were loaded. Additional SDS-PAGE analyses were performed for egg supernatants collected on days 0, 7, 14, and 21 using gels of the same proportions.

2.4. Protein Bands Cutting and Protein Re-Collection

To investigate protein changes during embryonic development, prominent protein bands from day-zero eggs were excised and analyzed using the Bradford Protein Assay Kit (Beyotime Biotechnology, Shanghai, China). These samples underwent qualitative analysis via LC-MS/MS following a standardized protocol.

2.5. LC-MS/MS for First-Day Eggs and Re-Collected Proteins from Bands

Protein digestion was performed for eggs at different incubation days and for proteins collected on the first day using the FASP method [30]. For each sample, 200 μg of protein underwent ultrafiltration (Pall, 10 kD) to remove detergents, DTT, and other low-molecular-weight contaminants. The samples were repeatedly centrifuged with 200 μL of UA buffer (8 M urea, 150 mM Tris-HCl, pH 8.0). Following impurity removal, 100 μL of 0.05 M iodoacetamide in UA buffer was added to block reduced cysteine residues, and samples were incubated in the dark for 20 min. The filters were washed three times with 100 μL of UA buffer and twice with 100 μL of 25 mM NH4HCO3. Proteins were then digested overnight at 37 °C with 3 μg of trypsin (Promega, Madison, WI, USA) in 40 μL of 25 mM NH4HCO3, and the resulting peptides were collected.

2.6. Chromatographic Fractionation

Chromatographic separation was carried out using a column (0.15 mm × 150 mm, RP-C18, Column Technology Inc., Lombard, IL, USA) equilibrated with 95% buffer A (0.1% formic acid in water). Peptides were loaded onto Zorbax 300SB-C18 peptide traps (Agilent Technologies, Wilmington, DE, USA) and eluted with buffer B (0.1% formic acid, 84% acetonitrile [ACN]) using a linear gradient: 4% to 50% buffer B over 50 min, followed by 50% to 100% buffer B over 5 min, and held at 100% for 6 min.
The peptide eluates were analyzed using a Q Exactive mass spectrometer. The survey scan ranged from m/z 300 to 1800, capturing positive ions. For MS1, the resolution was set at 70,000 (at m/z 200), the AGC target was 3 × 106, the maximum injection time was 10 ms, and the dynamic exclusion was 40 s. Each scan selected 10 fragment ions for MS/MS, using higher-energy collisional dissociation (HCD) with a normalized collision energy of 30 eV and an under-fill ratio of 0.1%. The entire experiment was conducted in triplicate to ensure reproducibility.

2.7. PRM-Dynamic Changes in Forty Egg Proteins During Egg Development

The stable isotope iRT KIT peptide (Biognosys AG, Zurich, Switzerland) was added to each digested peptide sample across different incubation times, with three replicates per condition, serving as internal standards. Samples (2 μg in 40 μL of 0.1% formic acid buffer) underwent desalination using stage-tip-mounted C18 cartridges (Sigma-Aldrich, St. Louis, MO, USA) before reversed-phase chromatography on an nLC-1200 system (Thermo Fisher Scientific, Waltham, MA, USA). Liquid chromatography (LC) was performed with gradients of 5% to 35% ACN over 45 min. PRM analysis was conducted on a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA).
The full scan was performed with a 60 min analysis time in positive ion mode, covering a scan range of 300–1800 m/z, at a resolution of 60,000 (@m/z 200), with an AGC target of 3 × 106 and a maximum injection time of 200 ms. This was followed by a PRM event using an isolation window of 1.6 Th, a resolution of 30,000 (@m/z 200), an AGC target of 3 × 106, and a maximum injection time of 120 ms. HCD was employed for MS2 activation, with a normalized collision energy set at 27 eV. Raw data analysis was conducted using Skyline (MacCoss Lab, University of Washington, Seattle, WA, USA), with peptide signal intensity quantified based on the peak area for each sample and normalized to internal standards.

2.8. MS Data Analysis

Raw data files were searched against the H. hystricis transcriptome using Mascot (version 2.2), employing trypsin as the enzyme with up to two allowed missed cleavages. Carbamidomethylation was set as a fixed modification for cysteines, while N-terminal acetylation and methionine oxidation were treated as variable modifications. The decoy database was set to reverse, with peptide and fragment mass tolerances set at ±20 ppm and 0.1 Da, respectively, and a Mascot score threshold of ≥20. A global false discovery rate of 0.01 was applied for peptide and protein identification. Identified polypeptides/proteins were further queried against a custom H. hystricis protein database compiled from transcriptomic data (NCBI: PRJNA1168713), and homologous protein searches were conducted using the UniProt database (https://www.uniprot.org/, accessed on 27 November 2024).
Label-free quantification was carried out using MaxQuant (version 1.6.14.0, https://maxquant.net/maxquant/, accessed on 27 November 2024) according to Tyanova et al. [31], with protein abundance assessed by intensity-based absolute quantification (iBAQ). Protein levels were determined based on normalized spectral intensities.

3. Results and Discussion

Ticks pose a considerable threat to a wide range of terrestrial vertebrates, including mammals, birds, reptiles, and amphibians. As obligate hematophagous parasites, they cause adverse effects such as anemia, malnutrition, and damage to the host’s integument. They are also vectors for various pathogens, including fungi, viruses, and bacteria (e.g., rickettsiae), leading to diseases like Lyme disease, Rocky Mountain spotted fever, and Mediterranean spotted fever. Given the need for sustainable tick control strategies, vaccination-based approaches are recognized as more environmentally friendly compared to chemical methods. Since tick eggs contain all the essential components for embryonic development, they may act as reservoirs for protective antigens, making the targeting of proteins during the egg incubation phase a promising strategy for tick control.

3.1. Protein Expression Profile at Different Egg Developmental Stages

As shown in Figure 1, SDS-PAGE analysis revealed that tick egg proteins ranged in size from 15 to 250 kDa, with bands 3, 4, 5, and 6 being the most prominent. LC-MS/MS analysis of day-one egg samples identified vitellogenins (Vgs) as the predominant proteins in these high-intensity bands (Figure 2). Protein band intensity remained relatively unchanged across different incubation days for bands 3, 4, 5, and 6, while gradual reduction was observed for bands 1, 2, 8, 10, and 11. Notably, bands 7 and 12 showed reduced intensity by the 14th day of incubation, whereas band 9 exhibited increased intensity at day 7.
To identify the proteins in these bands, all 12 bands were excised, and the proteins were extracted using a Protein Extraction Kit (Bio-Rad, Hercules, CA, USA). The re-collected proteins were analyzed via LC-MS/MS, and after annotation, they were ranked according to their iBAQ values. The results revealed that 9 out of the 12 bands predominantly contained vitellogenin (Cluster-17602.25911) and vitellin-a (Cluster-17602.43418), indicating that vitellogenin serves as the primary nutrient source in day-one eggs. Furthermore, these results suggest that the ovary may directly absorb exogenous vitellogenin from the adult female tick. This is supported by the work of Yang et al., who demonstrated that a process of protein auto-synthesis occurs during the early stages of ovarian development, with developing oocytes absorbing vitellogenin from the hemolymph of the adult female tick.
Other prominent proteins identified included actin 1 (Cluster-17602.36441), Kunitz domain-containing protein 1 (Cluster-17602.30986), yolk protein (Cluster-17602.15229), and cathepsin D (Cluster-17602.33592). Additionally, minor proteins such as histone H4 (Cluster-17602.34926), cysteine-rich protein (Cluster-17602.29432), and serpin (Cluster-11893.0) were also detected (Figure 2). These proteins are known to participate in processes like anti-inflammation, anti-hemostasis, and immune responses in adult ticks, though their roles in tick eggs remain to be clarified.

3.2. Identification of High-Confidence Polypeptides and Their Dynamics Changes in Egg Development

The LC-MS/MS analysis of day-one tick egg samples searched against a protein library derived from the H. hystricis transcriptome and identified 1416 unique peptides and 207 polypeptides. Among these, 125 sequences were classified as high-confidence transcripts, with each unique peptide being matched at least twice. BLAST annotation against the tick database in UniProt revealed 108 transcripts, of which 43 sequences had over 80% identity with known proteins, 28 exhibited 60–80% identity, and 37 showed lower identity with previously released proteins.
The abundance of these high-confidence proteins, based on iBAQ, varied significantly. Notably, 16 proteins had iBAQ values exceeding 1.00E+10, indicating their high abundance in day-one egg samples (Table 1).
To further investigate the roles of these proteins during embryonic development, Parallel Reaction Monitoring (PRM), a highly sensitive and reproducible mass spectrometry quantification technique, was employed to measure the levels of 40 target proteins across four stages of egg incubation (0, 7, 14, and 21 days) (Supplementary Table S1). This approach allowed for precise monitoring of protein abundance changes throughout tick oogenesis.
The 40 selected proteins included 18 enzymes, five serpins, one Kunitz domain-containing protein, one α2-macroglobulin, two neutrophil elastase inhibitors (NEI), three heat shock protein 70 (HSP70), four vitellogenins (Vgs), three cytoskeletal proteins, one ferritin, one elongation factor, and one uncharacterized protein (Figure 3). These targets were chosen based on their potential importance in egg development and to observe their dynamic expression during the different stages of tick embryogenesis.

3.3. Classification and Potential Function of Egg Proteins

Based on their functions and families, the 108 transcripts identified were classified into eight categories: transporters (20), enzymes (28), immunity and antimicrobial-related proteins (7), proteinase inhibitors (20), cytoskeletal proteins (5), heat shock proteins (3), secreted proteins (9), and uncharacterized proteins (16) (Supplementary Table S2). These categories play essential roles in processes critical to tick egg development, including nutrient supply, metabolism, molecular synthesis and transfer, as well as antimicrobial defense and immunity.
Among the 20 enzymes identified were cysteine proteases, aspartic proteinases, hydrolases, dehydrogenases, and peroxidases. LC-MS/MS analysis of day-one eggs showed that only yolk cathepsin and heme-binding aspartic proteinase (Cluster-11877.0) had high abundances, with iBAQ values exceeding 1.00E+10. The remaining enzymes displayed generally lower iBAQ values. Given the substantial diversity of egg proteins and the complex changes occurring throughout egg development, these enzymes are discussed in separate groups to provide a more detailed analysis.

3.4. Vgs

Vg, a yolk protein precursor synthesized in the fat body of female ticks, is secreted into the hemolymph and absorbed by developing oocytes. Vitellin (Vt), derived from Vg, acts as the primary nutrient and energy source during embryogenesis, playing a pivotal role in tick egg development.
The analysis identified one Vt and five Vg proteins as major components in day-one eggs, with Vt (Cluster-17602.43418) and one Vg (Cluster-17602.25911) showing particularly high abundances (iBAQ > 1.00E+10), suggesting a dependence on Vg and Vt throughout egg development (Figure 2 and Figure 3A).
The presence of Vg and Vt across nine distinct bands, corresponding to molecular weights of approximately 16, 40, 45, 50, 63, 70, 110, 200, and 280 kDa, indicates that Vg undergoes digestion into various fragments. This observation supports the findings by Silveira et al. [32], who described Vg as a hemolymphatic phospholipoglycoprotein. Inside the oocyte, Vg is cleaved to produce Vt, consisting of nine polypeptides with molecular weights of 203, 147, 126, 82, 74, 70, 61, 47, and 31 kDa [33]. Thus, Vt is likely generated from Vg during the incubation period. This dynamic relationship is further supported by LC-MS/MS data (Figure 2), showing that the band intensities of Vg and Vt remain relatively stable during development, as seen in bands 3, 4, 5, and 6 in Figure 1. The consistent presence suggests that Vg degradation is an ongoing process, with continuous conversion to Vt throughout incubation. For instance, band 9 showed increased intensity on day 7, indicating that one of the increasing proteins may be Vt, initially detected on day 0, accounting for only 13% of the total protein.
Considering Vg’s critical role in egg development, PRM was employed to examine its concentration variations across different developmental stages. The PRM results showed that Vg levels fluctuated 1-4-fold over the four incubation stages (Figure 3A), suggesting that Vg is increasingly digested into Vt to sustain the demands of egg development.

3.5. Lipocalins

Lipocalins are low-molecular-weight proteins primarily responsible for transporting small molecules [29]. In soft ticks, the lipocalin family is notably abundant in terms of both the number of members and expression levels [34,35,36,37,38]. This study identified 13 lipocalin-annotated transcripts in day-one tick eggs, with sequence identities ranging from 40% to 70% compared to other tick species, indicating considerable sequence variability among lipocalins [39].
While lipocalins are known to interact with histamine, serotonin, and leukotriene B4 to reduce host inflammation in ticks [40,41,42] and are predominantly expressed in salivary glands and secreted into saliva [14,35,36], their role in tick eggs appears different. The identified lipocalins were present at low levels in day-one eggs, suggesting that their function may involve the transport of nutritional small molecules or other processes supporting embryonic differentiation and growth [40] rather than the primary anti-inflammatory role observed in adult tick saliva [43,44,45,46,47].
Despite limited knowledge about lipocalin function in tick eggs, one study on recombinant lipocalin (rHlLIP) from Haemaphysalis longicornis has shown its ability to reduce tick hatch rates, indicating a potentially critical role in tick development [48]. Therefore, further research is necessary to elucidate the specific functions of lipocalins during tick embryogenesis.

3.6. Cysteine Proteases

Proteases are vital for protein hydrolysis in organisms and are categorized based on their catalytic mechanisms into serine, cysteine, aspartic, threonine, and metalloproteases. This study identified four cysteine proteases and six aspartic proteases in day-one tick eggs, both of which play key roles in protein hydrolysis, particularly in the degradation of vitellin during embryonic development.
In parasitic organisms, cysteine proteases belong to the papain family and are divided into two major groups: clan CA [49] and clan CD [50]. These enzymes are involved in various biological processes, such as protein degradation and digestion [51].
Among the identified cysteine protease sequences, three showed 78%, 82.2%, and 92.4% identity with known tick midgut cysteine proteases, though their abundance in day-one eggs was low. Given the substantial amount of vitellin or yolk protein needed for egg development, a single cysteine protease is insufficient for complete digestion, suggesting that additional proteases from this family or related families contribute to nutrient processing. For instance, cathepsin L, a cysteine protease known for its potent haemoglobinase activity [52,53,54], plays a critical role in the hydrolysis of vitellin [20,55,56]. In this study, cathepsin L (Cluster-17602.36868) remained relatively stable during the first three stages of egg development, but its quantity increased significantly by 1.65-fold on day 21, as indicated by PRM results (Figure 3J). Notably, cathepsin was the main component of Band 7, constituting 81% of its composition (Figure 2). By day 14, Band 7 showed a marked reduction in protein abundance (Figure 1), suggesting that cathepsin levels decrease in later stages, potentially due to reduced demand for cathepsin activity as other proteases take over roles in preparation for larval hatching. The identification of a diverse array of proteases in tick eggs indicates the presence of a complex digestive cascade that facilitates nutrient digestion during embryogenesis.

3.7. Aspartic Proteases

Similar to cysteine proteases, the aspartic proteinase family is also essential for protein digestion and represents a major enzymatic component in the tick gut [57]. The breakdown of hemoglobin in ticks relies on a network of cysteine and aspartic proteinases [52]. In this study, four aspartic proteinases and two cathepsin D proteins were identified, both belonging to the aspartic proteinase family. Like cysteine proteases, aspartic proteinases participate in vitellin hydrolysis, showing peak activity at a heme-to-globin ratio of 1:1, which suggests that vitellin degradation may be regulated based on heme availability [19].
Two heme-associated aspartic proteinases were detected, with PRM results indicating an increase in one heme-binding aspartic proteinase on day 21 of incubation, while the other remained stable throughout egg development. These variations in expression likely contribute to the diversity of hydrolytic activities within the aspartic proteinase family. Previous research has shown that aspartic proteinase precursor (BYC) exhibits a slow rate of vitellin hydrolysis [58] due to the absence of a critical aspartic acid residue, in contrast to the egg cysteine endopeptidase (VTDCE), which demonstrates higher hydrolytic activity toward vitellin [59].
Cathepsin D, a key lysosomal aspartic protease, exhibited a slight decline in abundance during egg development according to PRM analysis, with both identified isoforms (Cluster-18960.0 and Cluster-17602.1417) showing reduced levels as development progressed (Figure 3B). This finding is consistent with findings by [60], which noted a gradual decrease in cathepsin D activity after initial egg incubation. This trend contrasts with bisexual H. longicornis, where cathepsin D expression and activity peak on days 11 and 13, respectively [61].
The fluctuating levels of cathepsin expression and activity across tick species suggest that cathepsin D may have a stage-specific regulatory role in egg development. In our study, cathepsin D (Cluster-17602.33592) was a major component of bands 8 (69%) and 9 (8%), with band 8 showing a slight decline in protein abundance after day 14 of incubation (Figure 2). This decline may indicate a reduced demand for cathepsins in the later stages of egg development, potentially compensated by other cysteine and aspartic proteases, reinforcing the hypothesis that a complex digestive cascade is present in tick eggs.

3.8. GSTs

During blood feeding, ticks are exposed to elevated levels of reactive oxygen species (ROS) due to the host blood’s high iron content. As ticks concentrate the ingested blood, iron levels increase, which can react with oxygen in the tick’s body, leading to the formation of ROS, such as hydrogen peroxide (H2O2) [62]. H2O2 poses a significant threat to aerobic organisms by causing extensive damage to membrane lipids, nucleic acids, and proteins [63].
To mitigate the harmful effects of ROS and ensure survival, ticks employ both enzymatic and non-enzymatic mechanisms to reduce oxidative damage. Key detoxification enzymes, such as catalase, glutathione S-transferases (GSTs), and glutathione peroxidase, catalyze the conversion of ROS into less harmful molecules.
In this study, four sequences related to enzymatic detoxification were identified, including one (Cluster-17602.35954) showing 78% identity with GST. GSTs play a critical role in managing chemical toxicity and environmental stress, particularly in response to insecticide exposure. Although LC-MS/MS results indicated that GST was not a predominant protein on the first day of egg incubation, PRM analysis showed a marked increase in its expression during the late incubation stages, with a 4.12-fold rise by day 21 (Figure 3C). This pattern aligns with findings in Boophilus microplus [22], where GST levels increased progressively during embryonic development, peaking on day 20, which corresponded with the highest GST activity observed during this period. Knockdown experiments have shown that reduced GST expression increases susceptibility to insecticides in both larvae and adult male ticks, leading to higher mortality rates, decreased egg-laying capacity, and lower egg weight [64,65]. These results indicate that elevated GST levels are essential for successful larval hatching.
Beyond detoxification, GSTs are also involved in transporting endogenous hydrophobic compounds, such as hormones, steroids, and hemoglobin [66], and they play a role in viral defense mechanisms [67]. This multifunctionality underscores the significance of GSTs in tick eggs, suggesting they contribute to various physiological processes essential for embryonic development and survival.

3.9. Catalase

Unlike GSTs, which are involved in detoxification, catalase plays a key role in reducing oxidative stress in ticks by breaking down H2O2 [22]. H2O2 is generated in the tick midgut, where high concentrations of hemoglobin, iron, and oxygen are present. In tick eggs, two catalase-related sequences, Cluster-17602.16856 and Cluster-17602.16862, have been identified, exhibiting 73.2% and 73.9% identity with catalase, respectively. The abundance of these sequences increased during egg development, with Cluster-17602.16862 showing a marked surge on day 21 (Figure 3C), indicating elevated H2O2 levels and rising oxidative stress throughout the developmental stages.
To mitigate the toxicity of H2O2, ticks upregulate antioxidant production to combat oxidative damage, thereby ensuring proper egg development and hatching. This mechanism is consistent with observations in other organisms, where catalase catalyzes the conversion of H2O2 into water and oxygen [68]. The essential role of catalase in egg development is further supported by Kumar et al. [69], who found that silencing the catalase gene resulted in decreased egg weight and reduced hatching rates.
Catalase is also implicated in transovarial virus transmission. Its gradual increase during egg development may help limit viral proliferation. Budachetri et al. [70] reported a 2- to 11-fold rise in catalase expression upon viral infection and observed a 44% reduction in transovarial virus transmission following catalase knockdown

3.10. Peroxiredoxins(Prx)

Prx also functions as a H2O2 scavenger [71]. Studies demonstrate that Prx, which possesses conserved cysteine residues, efficiently neutralizes H2O2 [72]. One identified sequence showed 96.6% identity with H. longicornis Prx, confirming the annotation. In H. longicornis, Prx (Cluster-17602.35118) exhibited a 2-fold increase just before larval hatching (Figure 3C), likely due to elevated lipid peroxidation and rising oxygen consumption during late egg development Freitas et al. [22]. The upregulation of Prx is essential for successful egg development and hatching [72]. Prx knockdown experiments revealed elevated H2O2 levels in Ixodes scapularis cell lines treated with paraquat, highlighting its vital role in managing oxidative stress [73].
Similar to catalase, Prx is associated with virus transmission, although one form of Prx has been suggested to facilitate viral replication through mechanisms beyond H2O2 clearance [74]. These findings emphasize the multifunctional roles of antioxidant enzymes in tick eggs, underscoring their importance in oxidative stress management and potentially influencing viral dynamics.

3.11. Dehydrogenase

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a critical enzyme in the glycolytic pathway, the primary metabolic route for energy production from glucose.
One GAPDH was identified in day-one tick eggs, with levels steadily increasing throughout incubation, indicating a growing need for GAPDH to meet the energy demands of egg development. This observation aligns with findings by Hildebrandt et al. [75], which reported that GAPDH catalyzes the conversion of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate, thereby facilitating ATP production.
Additionally, aldehyde dehydrogenase (Cluster-17602.38580), another relevant enzyme, showed an 11-fold increase in expression by day 21 of egg development (Figure 3D). Although its role in ticks remains poorly understood, the significant upregulation suggests it plays a pivotal role in supporting egg development.

3.12. Hydrolases

Nine hydrolases were detected in day-one eggs, with five selected for further investigation using PRM to monitor dynamic changes during development. An upward trend was observed in three hydrolases: neurotrypsin (Cluster-17602.21941), ATP synthase subunit beta (Cluster-17602.34511), and alpha-mannosidase (Cluster-17602.36136).
Neurotrypsin levels surged 15-fold by day 7 compared to day-one levels, while ATP synthase subunit beta increased on day 7 and peaked on day 21 during the larval stage. Alpha-mannosidase exhibited a 2-fold rise by day 7, maintaining this level through day 21 (Figure 3E). In contrast, carboxypeptidase and phospholipase showed no significant variation from day-one levels. The upregulation of most hydrolases during egg development indicates an elevated requirement for nutrient digestion, reflecting the increasing metabolic demands associated with embryogenesis and preparation for hatching

3.13. AMP

Innate immunity is a fundamental aspect of tick biology that significantly impacts development. It is regulated by various proteins, particularly antimicrobial peptides (AMPs) and serpins.
AMPs are essential effector molecules in the tick immune system, capable of neutralizing pathogenic microorganisms and playing a key role in innate immune defense [76]. Numerous tick AMPs, such as microplusin, VTDCE, defDM, Hb98-114, and defensin, have been detected in tissues, including the salivary glands, hemolymph, and midgut [77,78,79,80].
In this study, several AMPs with antimicrobial activity were identified, including microplusin, alpha-2-macroglobulin protein (α2M), cysteine-rich protein, peroxinectin, and histidine-rich proteins. Cluster-17602.36041 showed an 80% similarity to α2M from Amblyomma cajennense, and proteomic analysis revealed an 11-fold and 15-fold increase in its levels at days 7 and 14, respectively, with a significant rise by day 21 compared to day-one eggs (Figure 3J). This upregulation suggests enhanced innate immune responses during late egg development, as α2M protects against harmful proteases from various sources, including pathogens [81]. Buresova et al. [82] demonstrated that silencing α2M in Ixodes ricinus through dsRNA interference reduced the phagocytic activity of hemocytes against Chryseobacterium indologenes, both in vitro and in vivo.
Cluster-17602.29432 exhibited high similarity to cysteine-rich proteins and a notably high iBAQ value of 1.11E+09 in day-one eggs, suggesting a significant role in innate immunity during early egg development. Cysteine-rich proteins are known to inhibit Gram-negative bacteria and proteolytic enzymes such as chymotrypsin and elastase [83,84].
In contrast, Clusters 17602.7590 and 17602.7506, which showed lower identity with microplusin and peroxinectin, also had lower iBAQ values. Microplusin, a cysteine-rich AMP, exerts its antibacterial effects through metal binding [85] but was present in lower quantities in day-one eggs. Similarly, peroxinectin levels were low in day-one eggs and remained underexplored beyond its initial identification in Rhipicephalus microplus [86]. These two proteins have not been analyzed through PRM trials, leaving their expression dynamics during egg development unknown. Considering the involvement of multiple proteins in tick egg innate immunity, further research is necessary to investigate the mechanisms and functional roles of these immunity-related proteins in this context. Additionally, two transcripts showed 38% and 40% identity with his-rich 1 fat body overexpressed and were categorized as immunity-related proteins. This classification is based on the presence of histidine-rich residues in many AMPs at their C- and N-termini [85,87]. Given the complexity of immunity-related proteins in tick eggs, further research is needed to elucidate the mechanisms underlying their roles in tick innate immunity during development.

3.14. Serpin

Proteinase inhibitors are categorized into at least 18 families based on their primary and tertiary structures and inhibition mechanisms [88]. In ticks, four main groups of serine protease inhibitors have been identified: Kunitz domain inhibitors, Kazal domain inhibitors, trypsin inhibitor-like cysteine-rich domain (TIL) inhibitors, and serpins [89].
Ticks are particularly rich in proteinase inhibitors, especially serpins. This study identified 20 sequences annotated as proteinase inhibitors in tick eggs, including eight serpins, five Kunitz domain-containing proteins, and six NEIs.
Seven of the sequences were classified as serpins, showing 54% to 94% identity. Two serpins, Cluster-17602.31952 and Cluster-11893, were highly abundant in day-one eggs, with iBAQ values of 1.18E+09 and 4.20E+09, respectively. LC-MS/MS analysis confirmed that these serpins were predominant in bands 7, 11, and 12, accounting for 5%, 5%, and 10% of the total protein content in these bands (Figure 2). While protein abundance in band 11 remained stable throughout the four stages of egg development, bands 7 and 12 showed a rapid decline in protein levels by day 14. This pattern suggests the primary proteins in these bands are being utilized, whereas the serpins are not, as evidenced by a three-fold increase in Cluster-17602.31952 abundance by day 21, according to PRM results (Figure 3F).
The high abundance and upregulation of these serpins during tick egg development imply a significant role in reproductive processes. A similar trend was reported in Rhipicephalus haemaphysaloides, where serpin RHS8 mRNA expression increased from the feeding stage to oviposition. RNA interference (RNAi) experiments demonstrated that serpin silencing led to reduced body weight, extended feeding duration, fewer eggs laid, and lower egg hatchability [90,91], underscoring the critical role of serpins in tick reproduction.
Serpins are well-established anti-inflammatory proteins in adult ticks, known for their ability to modulate the host’s immune response by influencing immune cell migration to sites of inflammation [92]. However, tick eggs do not encounter the host’s immune system. It is hypothesized that components of the host’s blood transferred to the eggs may not be completely metabolized. In tick eggs, serpins might serve a similar function as in adults, providing anti-inflammatory effects that support development and hatching. This mechanism likely involves interactions with proteases such as serine protease, cathepsin G, thrombin, and chymotrypsin. For example, RmS-15 inhibits thrombin activity [93], while Amblyomma americanum serpin AAS19 inhibits both cathepsin G and thrombin [94].
Additionally, serpins act as anticoagulants by suppressing coagulation factors [94] or by blocking the intrinsic and common pathways of the coagulation cascade [95]. Noteworthy examples include serpins such as Iris [96], Iripin-8 [95], and AAS19 [97], which inhibit coagulation factors like FXa, FVIIa, and FIXa.
As egg development progresses, particularly during the late stages when larvae are about to hatch, the demand for serpins may increase to prevent hemolymph coagulation in the emerging larvae. Proper regulation of coagulation is critical for successful hatching and survival, as it can prevent blockages that could hinder larval development. Understanding the specific functions of serpins during this critical phase could provide valuable insights into tick physiology and adaptive strategies in response to environmental pressures.
Serpins are also associated with anti-complement activities [98,99,100,101] and may facilitate viral colonization [102]. However, whether serpins play a role in preventing the transovarial transmission of pathogens during egg development remains uncertain.

3.15. Kunitz Domain-Containing Protein

The Kunitz domain-containing protein, known for its thrombin-inhibiting activity, is part of the serpin family. In this study, five Kunitz domain-containing proteins exhibited low sequence identity (35–46%) compared to those from Rhipicephalus microplus. PRM results indicated that one Kunitz protein (Cluster-17602.19391) was downregulated during the egg protein stage (Figure 3F). SDS-PAGE and LC-MS/MS analyses identified another Kunitz protein (Cluster-17602.30986) as prominent in bands 10, 11, and 12, constituting 61%, 64%, and 9% of these bands, respectively (Figure 2). As egg development progressed, the abundance of these bands decreased, suggesting digestion of the primary Kunitz domain-containing proteins. The potential function of Kunitz proteins during egg development remains unclear in the current study, despite the extensive study of other Kunitz proteins for anticoagulant properties, such as BmTI from Boophilus microplus, Doenitin-1 from Haemaphysalis doenitzi [103], Boophilin from Rhipicephalus microplus [104,105], and Hemalin from H. longicornis [106]. Kunitz proteins may be involved in anticoagulation in tick eggs, but the reason for the decrease in Kunitz abundance remains unclear. It is possible that different Kunitz family members take on anticoagulant roles at various stages of egg development. In this context, further investigation into the functional roles of the Kunitz protein is warranted.

3.16. Neutrophil Elastase Inhibitors (NEI)

More than half of the immune-related proteins identified in this study were NEIs, with iBAQ values indicating high abundance: three exceeded 1.00E+10, two ranged between 100 and 1000 (including Cluster-17602.36134 and Cluster-17602.34822), and one was below 100 (Cluster-17602.33042). Notably, the expression levels of two NEIs (Cluster-17602.36134 and Cluster-17602.33042) increased by 1.3- to 1.8-fold in day-21 eggs, suggesting their involvement in egg development (Figure 3G).
The high abundance of NEIs in tick eggs implies a significant role in immunity. NEIs, which are enzymes produced by host immune cells, degrade proteins as part of the defense against pathogens and may inhibit neutrophil elastase activity [107], potentially enhancing defenses against parasitic threats. The immune function of NEIs has also been observed in the eggs of the blood-feeding insect Triatoma infestans [108].
NEIs belong to the pacifastin family, which is part of the conserved serpin superfamily in arthropods [108,109], indicating functional similarities with other serpins. However, their specific roles in ticks are still largely unexplored. Further research is required to fully elucidate the potential contributions of NEIs to tick development and immune regulation.

3.17. HSP

The heat shock protein (HSP) family plays a critical role in helping cells and organisms cope with high temperatures and various stress conditions, including exposure to toxins and pathogen infections. Numerous heat shock proteins have been reported in insects [110,111,112,113,114]. In ticks, HSP70, a prominent member of this family, has been linked to pathogen infections and exhibits anticoagulant activity [115,116,117]. Our findings indicate that HSP70 expression is upregulated during tick egg development (Figure 3H), suggesting that increased levels are necessary as hatching approaches. However, the specific functions of HSP70 in tick eggs remain poorly understood.

3.18. Cytoskeletal Protein and Other Proteins

Among cytoskeletal proteins, a significant upregulation (4-6-fold) was observed by day 14, implying an increased demand for these proteins to support larval growth, particularly as tissue formation begins around day 7 [118] (Figure 3I). Within the groups of secreted and uncharacterized proteins, three were found to be highly abundant in day-one eggs, though their precise identities remain undetermined. PRM analysis of 40 proteins revealed that one uncharacterized protein (Cluster-17602.39905) showed a dramatic 43-fold increase by day 21 compared to day one (Figure 3J). Additionally, Elongation Factor 1-alpha (Cluster-17602.36724) exhibited the most significant rise, with a 292-fold increase by day 21 (Figure 3J). Despite limited insights into their functions in ticks, the substantial abundance of these proteins suggests they play critical roles in egg development, warranting further functional studies.

4. Conclusions

This study identified a broad range of proteins in tick eggs, with transporters, enzymes, and proteinase inhibitors accounting for half of the total. These proteins are likely involved in nutrient supply, detoxification, protein digestion, and innate immunity. The extensive diversity and dynamic changes in the proteome of H. hystricis eggs, from day one of incubation to day 21, may aid in identifying new targets for vaccine development, for example, catalase, cysteine protease and aspartic protease, AMPs, etc. They are important for egg development. Therefore, tracking their dynamic changes during embryonic development can be a key research direction for further studies, such as RNA interference and protein expression in vitro for anti-tick vaccine. By developing vaccines targeting these proteins, it may be possible to interfere with egg development, thereby suppressing tick population growth and reducing their ability to transmit pathogens. Though these proteins are not directly exposed to the environment during the tick lifecycle, they remain promising vaccine candidates.
The specific roles of some egg proteins remain unclear, such as Kunitz domain-containing proteins, dehydrogenases, and hydrolases, but this research offers a comprehensive overview of egg proteins and lays the foundation for future studies.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ani14233466/s1, Table S1. Top four proteins identified from the LC-MS/MS analysis of protein bands from day-one egg samples. Table S2. High-confidence transtripts identified in eggs of H. hystricis on day-one incubation.

Author Contributions

Conceptualization: Q.T. and T.C.; writing—original draft: Q.T.; review and editing: W.L. All authors have read and agreed to the published version of the manuscript.

Funding

This study was funded by the project of Hunan Agricultural University (Changsha, China, 2019xny-js053).

Institutional Review Board Statement

The collection process was approved by the Hunan Provincial Department of Animal Protection and supervised by the Hunan Animal Health and Usage Committee (No. 43321503), ensuring no harm to the animals.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original dataset generated and analyzed during the current study is included in the Supplementary Materials.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Chao, L.L.; Hsieh, C.K.; Ho, T.Y.; Shih, C.M. First zootiological survey of hard ticks (Acari: Ixodidae) infesting dogs in northern Taiwan. Exp. Appl. Acarol. 2019, 77, 105–115. [Google Scholar] [CrossRef] [PubMed]
  2. Yamauchi, T.; Yano, S.; Yamamoto, T.; Yamamoto, E.; Miyamoto, T. Ticks (Acari: Ixodidae) from medium-sized to large mammals in Ehime Prefecture, Japan. Exp. Appl. Acarol. 2013, 60, 263–270. [Google Scholar] [CrossRef] [PubMed]
  3. Khoo, J.J.; Lim, F.S.; Tan, K.K.; Chen, F.S.; Phoon, W.H.; Khor, C.S.; Pike, B.L.; Chang, L.Y.; AbuBakar, S. Detection in Malaysia of a Borrelia sp. From Haemaphysalis hystricis (Ixodida: Ixodidae). J. Med. Entomol. 2017, 54, 1444–1448. [Google Scholar] [CrossRef] [PubMed]
  4. Bezerra-Santos, M.A.; de Macedo, L.O.; Nguyen, V.L.; Manoj, R.R.; Laidoudi, Y.; Latrofa, M.S.; Beugnet, F.; Otranto, D. Cercopithifilaria spp. in ticks of companion animals from Asia: New putative hosts and vectors. Ticks Tick Borne Dis. 2022, 13, 101957. [Google Scholar] [CrossRef] [PubMed]
  5. Jongejan, F.; Su, B.L.; Yang, H.J.; Berger, L.; Bevers, J.; Liu, P.C.; Fang, J.C.; Cheng, Y.W.; Kraakman, C.; Plaxton, N. Molecular evidence for the transovarial passage of Babesia gibsoni in Haemaphysalis hystricis (Acari: Ixodidae) ticks from Taiwan: A novel vector for canine babesiosis. Parasit. Vectors 2018, 11, 134. [Google Scholar] [CrossRef]
  6. Lu, M.; Tian, J.H.; Yu, B.; Guo, W.P.; Holmes, E.C.; Zhang, Y.Z. Extensive diversity of rickettsiales bacteria in ticks from Wuhan, China. Ticks Tick Borne Dis. 2017, 8, 574–580. [Google Scholar] [CrossRef]
  7. Zhang, X.; Geng, J.; Du, J.; Wang, Y.; Qian, W.; Zheng, A.; Zou, Z. Molecular Identification of Rickettsia Species in Haemaphysalis Ticks Collected from Southwest China. Vector Borne Zoonotic Dis. 2018, 18, 663–668. [Google Scholar] [CrossRef]
  8. Lim, F.S.; Khoo, J.J.; Tan, K.K.; Zainal, N.; Loong, S.K.; Khor, C.S.; AbuBakar, S. Bacterial communities in Haemaphysalis, Dermacentor and Amblyomma ticks collected from wild boar of an Orang Asli Community in Malaysia. Ticks Tick Borne Dis. 2020, 11, 101352. [Google Scholar] [CrossRef]
  9. Khatri-Chhetri, R.; Wang, H.C.; Chen, C.C.; Shih, H.C.; Liao, H.C.; Sun, C.M.; Khatri-Chhetri, N.; Wu, H.Y.; Pei, K.J. Surveillance of ticks and associated pathogens in free-ranging Formosan pangolins (Manis pentadactyla pentadactyla). Ticks Tick Borne Dis. 2016, 7, 1238–1244. [Google Scholar] [CrossRef]
  10. Liu, Y.; Wang, L.; Wang, L.; Deng, L.; Wei, M.; Wu, K.; Huang, S.; Li, G.; Huang, Y.; Zhang, H.; et al. Characterization of the complete mitogenome sequence of the giant panda tick Haemaphysalis hystricis. Mitochondrial DNA B Resour. 2020, 5, 1191–1193. [Google Scholar] [CrossRef]
  11. Thekisoe, O.M.; Honda, T.; Fujita, H.; Battsetseg, B.; Hatta, T.; Fujisaki, K.; Sugimoto, C.; Inoue, N. A trypanosome species isolated from naturally infected Haemaphysalis hystricis ticks in Kagoshima Prefecture, Japan. Parasitology 2007, 134, 967–974. [Google Scholar] [CrossRef] [PubMed]
  12. Takada, N.; Fujita, H.; Yano, Y.; Oikawa, Y.; Mahara, F. Vectors of Japanese spotted fever. Jpn. J. Infect. Dis. 1992, 66, 1218–1225, (In Japanese with English Summary). [Google Scholar]
  13. Chiang, P.S.; Lai, Y.W.; Chung, H.H.; Chia, Y.T.; Wang, C.C.; Teng, H.J.; Chen, S.L. First molecular detection of a novel Babesia species from Haemaphysalis hystricis in Taiwan. Ticks Tick Borne Dis. 2024, 15, 102284. [Google Scholar] [CrossRef] [PubMed]
  14. Feng, L.L.; Cheng, T.Y. A survey of proteins in midgut contents of the tick, Haemaphysalis flava, by proteome and transcriptome analysis. Exp. Appl. Acarol. 2020, 80, 269–287. [Google Scholar] [CrossRef] [PubMed]
  15. Liu, L.; Cheng, T.Y.; He, X.M. Proteomic profiling of the midgut contents of Haemaphysalis flava. Ticks Tick Borne Dis. 2018, 9, 490–495. [Google Scholar] [CrossRef] [PubMed]
  16. Liu, L.; Cheng, R.; Mao, S.Q.; Duan, D.Y.; Feng, L.L.; Cheng, T.Y. Saliva proteome of partially- and fully-engorged adult female Haemaphysalis flava ticks. Vet. Parasitol. 2023, 318, 109933. [Google Scholar] [CrossRef]
  17. Cheng, R.; Li, D.; Duan, D.Y.; Parry, R.; Cheng, T.Y.; Liu, L. Egg protein profile and dynamics during embryogenesis in Haemaphysalis flava ticks. Ticks Tick Borne Dis. 2023, 14, 102180. [Google Scholar] [CrossRef]
  18. Li, Y.; Cheng, R.; Liu, X.Y.; Mihaljica, D.; Cheng, T.Y. The effect of feeding on different hosts on the egg proteins in Haemaphysalis qinghaiensis tick. Parasitol. Res. 2024, 123, 197. [Google Scholar] [CrossRef]
  19. Sorgine, M.H.; Logullo, C.; Zingali, R.B.; Paiva-Silva, G.O.; Juliano, L.; Oliveira, P.L. A heme-binding aspartic proteinase from the eggs of the hard tick Boophilus microplus. J. Biol. Chem. 2000, 275, 28659–28665. [Google Scholar] [CrossRef]
  20. Fagotto, F. Yolk degradation in tick eggs: I. Occurrence of a cathepsin L-like acid proteinase in yolk spheres. Arch. Insect Biochem. Physiol. 1990, 14, 217–235. [Google Scholar] [CrossRef]
  21. Logullo, C.; Vaz Ida, S.; Sorgine, M.H.; Paiva-Silva, G.O.; Faria, F.S.; Zingali, R.B.; De Lima, M.F.; Abreu, L.; Oliveira, E.F.; Alves, E.W.; et al. Isolation of an aspartic proteinase precursor from the egg of a hard tick, Boophilus microplus. Parasitology 1998, 116 Pt 6, 525–532. [Google Scholar] [CrossRef] [PubMed]
  22. Freitas, D.R.; Rosa, R.M.; Moraes, J.; Campos, E.; Logullo, C.; Da Silva Vaz, I., Jr.; Masuda, A. Relationship between glutathione S-transferase, catalase, oxygen consumption, lipid peroxidation and oxidative stress in eggs and larvae of Boophilus microplus (Acarina: Ixodidae). Comp. Biochem. Physiol. A Mol. Integr. Physiol. 2007, 146, 688–694. [Google Scholar] [CrossRef] [PubMed]
  23. Willadsen, P.; McKenna, R.V. Trypsin-chymotrypsin inhibitors from the tick, Boophilus microplus. Aust. J. Exp. Biol. Med. Sci. 1983, 61 Pt 2, 231–238. [Google Scholar] [CrossRef] [PubMed]
  24. Esteves, E.; Fogaça, A.C.; Maldonado, R.; Silva, F.D.; Manso, P.P.; Pelajo-Machado, M.; Valle, D.; Daffre, S. Antimicrobial activity in the tick Rhipicephalus (Boophilus) microplus eggs: Cellular localization and temporal expression of microplusin during oogenesis and embryogenesis. Dev. Comp. Immunol. 2009, 33, 913–919. [Google Scholar] [CrossRef]
  25. Liu, Y.K.; Wang, A.B.; Liu, G.H.; Liu, L.; Cheng, T.Y.; Duan, D.Y. Morphological and molecular biological identification of Haemaphysalis hystricis. Chin. Vet. Sci. 2022, 50, 214–222. [Google Scholar] [CrossRef]
  26. Ernieenor, F.C.L.; Ernna, G.; Mariana, A. Phenotypic and genotypic identification of hard ticks of the genus Haemaphysalis (Acari: Ixodidae) in Peninsular Malaysia. Exp. Appl. Acarol. 2017, 71, 387–400. [Google Scholar] [CrossRef] [PubMed]
  27. Arrieta, M.C.; Leskiw, B.K.; Kaufman, W.R. Antimicrobial activity in the egg wax of the African cattle tick Amblyomma hebraeum (Acari: Ixodidae). Exp. Appl. Acarol. 2006, 39, 297–313. [Google Scholar] [CrossRef]
  28. Jiang, Z. Biology of Haemaphysalis Hystricis Supino. Acta Entomol. Sin. 1983, 26, 413–418. [Google Scholar]
  29. Fujisaki, K.; Kitaoka, S.; Morii, T. Comparative observations on some bionomics of Japanese ixodid ticks under laboratory cultural conditions. Natl. Inst. Anim. Health Q. 1976, 16, 122–128. [Google Scholar]
  30. Wiśniewski, J.R.; Zougman, A.; Mann, M. Combination of FASP and StageTip-based fractionation allows in-depth analysis of the hippocampal membrane proteome. J. Proteome Res. 2009, 8, 5674–5678. [Google Scholar] [CrossRef]
  31. Tyanova, S.; Temu, T.; Cox, J. The MaxQuant computational platform for mass spectrometry-based shotgun proteomics. Nat. Protoc. 2016, 11, 2301–2319. [Google Scholar] [CrossRef] [PubMed]
  32. Silveira, A.B.; Castro-Santos, J.; Senna, R.; Logullo, C.; Fialho, E.; Silva-Neto, M.A. Tick vitellin is dephosphorylated by a protein tyrosine phosphatase during egg development: Effect of dephosphorylation on VT proteolysis. Insect Biochem. Mol. Biol. 2006, 36, 200–209. [Google Scholar] [CrossRef] [PubMed]
  33. Yang, X.; Yu, Z.; He, Y.; Xu, X.; Gao, Z.; Wang, H.; Chen, J.; Liu, J. Purification of vitellin and dynamics of vitellogenesis in the parthenogenetic tick Haemaphysalis longicornis (Acari: Ixodidae). Exp. Appl. Acarol. 2015, 65, 377–388. [Google Scholar] [CrossRef]
  34. Mans, B.J.; Venter, J.D.; Vrey, P.J.; Louw, A.I.; Neitz, A.W. Identification of putative proteins involved in granule biogenesis of tick salivary glands. Electrophoresis 2001, 22, 1739–1746. [Google Scholar] [CrossRef]
  35. Mans, B.J.; Louw, A.I.; Neitz, A.W. The major tick salivary gland proteins and toxins from the soft tick, Ornithodoros savignyi, are part of the tick Lipocalin family: Implications for the origins of tick toxicoses. Mol. Biol. Evol. 2003, 20, 1158–1167. [Google Scholar] [CrossRef]
  36. Mans, B.J.; Andersen, J.F.; Francischetti, I.M.; Valenzuela, J.G.; Schwan, T.G.; Pham, V.M.; Garfield, M.K.; Hammer, C.H.; Ribeiro, J.M. Comparative sialomics between hard and soft ticks: Implications for the evolution of blood-feeding behavior. Insect Biochem. Mol. Biol. 2008, 38, 42–58. [Google Scholar] [CrossRef] [PubMed]
  37. Francischetti, I.M.; Mans, B.J.; Meng, Z.; Gudderra, N.; Veenstra, T.D.; Pham, V.M.; Ribeiro, J.M. An insight into the sialome of the soft tick, Ornithodorus parkeri. Insect Biochem. Mol. Biol. 2008, 38, 1–21. [Google Scholar] [CrossRef]
  38. Oleaga, A.; Escudero-Población, A.; Camafeita, E.; Pérez-Sánchez, R. A proteomic approach to the identification of salivary proteins from the argasid ticks Ornithodoros moubata and Ornithodoros erraticus. Insect Biochem. Mol. Biol. 2007, 37, 1149–1159. [Google Scholar] [CrossRef]
  39. Rodriguez-Valle, M.; Moolhuijzen, P.; Piper, E.K.; Weiss, O.; Vance, M.; Bellgard, M.; Lew-Tabor, A. Rhipicephalus microplus lipocalins (LRMs): Genomic identification and analysis of the bovine immune response using in silico predicted B and T cell epitopes. Int. J. Parasitol. 2013, 43, 739–752. [Google Scholar] [CrossRef]
  40. Neelakanta, G.; Sultana, H.; Sonenshine, D.E.; Andersen, J.F. Identification and characterization of a histamine-binding lipocalin-like molecule from the relapsing fever tick Ornithodoros turicata. Insect Mol. Biol. 2018, 27, 177–187. [Google Scholar] [CrossRef]
  41. Paesen, G.C.; Adams, P.L.; Nuttall, P.A.; Stuart, D.L. Tick histamine-binding proteins: Lipocalins with a second binding cavity. Biochim. Biophys. Acta 2000, 1482, 92–101. [Google Scholar] [CrossRef] [PubMed]
  42. Díaz-Martín, V.; Manzano-Román, R.; Siles-Lucas, M.; Oleaga, A.; Pérez-Sánchez, R. Cloning, characterization and diagnostic performance of the salivary lipocalin protein TSGP1 from Ornithodoros moubata. Vet. Parasitol. 2011, 178, 163–172. [Google Scholar] [CrossRef] [PubMed]
  43. Sangamnatdej, S.; Paesen, G.C.; Slovak, M.; Nuttall, P.A. A high affinity serotonin- and histamine-binding lipocalin from tick saliva. Insect Mol. Biol. 2002, 11, 79–86. [Google Scholar] [CrossRef] [PubMed]
  44. Beaufays, J.; Adam, B.; Decrem, Y.; Prévôt, P.P.; Santini, S.; Brasseur, R.; Brossard, M.; Lins, L.; Vanhamme, L.; Godfroid, E. Ixodes ricinus tick lipocalins: Identification, cloning, phylogenetic analysis and biochemical characterization. PLoS ONE 2008, 3, e3941. [Google Scholar] [CrossRef]
  45. Beaufays, J.; Adam, B.; Menten-Dedoyart, C.; Fievez, L.; Grosjean, A.; Decrem, Y.; Prévôt, P.P.; Santini, S.; Brasseur, R.; Brossard, M.; et al. Ir-LBP, an ixodes ricinus tick salivary LTB4-binding lipocalin, interferes with host neutrophil function. PLoS ONE 2008, 3, e3987. [Google Scholar] [CrossRef]
  46. Mans, B.J.; Ribeiro, J.M. Function, mechanism and evolution of the moubatin-clade of soft tick lipocalins. Insect Biochem. Mol. Biol. 2008, 38, 841–852. [Google Scholar] [CrossRef]
  47. Mans, B.J.; Ribeiro, J.M. A novel clade of cysteinyl leukotriene scavengers in soft ticks. Insect Biochem. Mol. Biol. 2008, 38, 862–870. [Google Scholar] [CrossRef]
  48. Wang, D.; Xu, X.; Lv, L.; Wu, P.; Dong, H.; Xiao, S.; Liu, J.; Hu, Y. Gene cloning, analysis and effect of a new lipocalin homologue from Haemaphysalis longicornis as a protective antigen for an anti-tick vaccine. Vet. Parasitol. 2021, 290, 109358. [Google Scholar] [CrossRef]
  49. Barrett, A.J. Classification of peptidases. Methods Enzymol. 1994, 244, 1–15. [Google Scholar] [CrossRef]
  50. Rawlings, N.D.; Barrett, A.J. Evolutionary families of peptidases. Biochem. J. 1993, 290 Pt 1, 205–218. [Google Scholar] [CrossRef]
  51. Sajid, M.; McKerrow, J.H. Cysteine proteases of parasitic organisms. Mol. Biochem. Parasitol. 2002, 120, 1–21. [Google Scholar] [CrossRef] [PubMed]
  52. Sojka, D.; Hajdusek, O.; Dvorák, J.; Sajid, M.; Franta, Z.; Schneider, E.L.; Craik, C.S.; Vancová, M.; Buresová, V.; Bogyo, M.; et al. IrAE: An asparaginyl endopeptidase (legumain) in the gut of the hard tick Ixodes ricinus. Int. J. Parasitol. 2007, 37, 713–724. [Google Scholar] [CrossRef] [PubMed]
  53. Horn, M.; Nussbaumerová, M.; Sanda, M.; Kovárová, Z.; Srba, J.; Franta, Z.; Sojka, D.; Bogyo, M.; Caffrey, C.R.; Kopácek, P.; et al. Hemoglobin digestion in blood-feeding ticks: Mapping a multipeptidase pathway by functional proteomics. Chem. Biol. 2009, 16, 1053–1063. [Google Scholar] [CrossRef] [PubMed]
  54. Franta, Z.; Sojka, D.; Frantova, H.; Dvorak, J.; Horn, M.; Srba, J.; Talacko, P.; Mares, M.; Schneider, E.; Craik, C.S.; et al. IrCL1—The haemoglobinolytic cathepsin L of the hard tick, Ixodes ricinus. Int. J. Parasitol. 2011, 41, 1253–1262. [Google Scholar] [CrossRef] [PubMed]
  55. Renard, G.; Garcia, J.F.; Cardoso, F.C.; Richter, M.F.; Sakanari, J.A.; Ozaki, L.S.; Termignoni, C.; Masuda, A. Cloning and functional expression of a Boophilus microplus cathepsin L-like enzyme. Insect Biochem. Mol. Biol. 2000, 30, 1017–1026. [Google Scholar] [CrossRef]
  56. Estrela, A.B.; Seixas, A.; Teixeira Vde, O.; Pinto, A.F.; Termignoni, C. Vitellin- and hemoglobin-digesting enzymes in Rhipicephalus (Boophilus) microplus larvae and females. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 2010, 157, 326–335. [Google Scholar] [CrossRef]
  57. Mendiola, J.; Alonso, M.; Marquetti, M.C.; Finlay, C. Boophilus microplus: Multiple proteolytic activities in the midgut. Exp. Parasitol. 1996, 82, 27–33. [Google Scholar] [CrossRef]
  58. Nascimento-Silva, M.C.; Leal, A.T.; Daffre, S.; Juliano, L.; da Silva Vaz, I., Jr.; Paiva-Silva Gde, O.; Oliveira, P.L.; Sorgine, M.H. BYC, an atypical aspartic endopeptidase from Rhipicephalus (Boophilus) microplus eggs. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 2008, 149, 599–607. [Google Scholar] [CrossRef]
  59. Seixas, A.; Leal, A.T.; Nascimento-Silva, M.C.; Masuda, A.; Termignoni, C.; da Silva Vaz, I., Jr. Vaccine potential of a tick vitellin-degrading enzyme (VTDCE). Vet. Immunol. Immunopathol. 2008, 124, 332–340. [Google Scholar] [CrossRef]
  60. Qiu, Z.X.; Li, Y.; Li, M.M.; Wang, W.Y.; Zhang, T.T.; Liu, J.Z. Investigation of three enzymes and their roles in the embryonic development of parthenogenetic Haemaphysalis longicornis. Parasit. Vectors 2020, 13, 46. [Google Scholar] [CrossRef]
  61. Zhang, T.T.; Qiu, Z.X.; Li, Y.; Wang, W.Y.; Li, M.M.; Guo, P.; Liu, J.Z. The mRNA expression and enzymatic activity of three enzymes during embryonic development of the hard tick Haemaphysalis longicornis. Parasit. Vectors 2019, 12, 96. [Google Scholar] [CrossRef] [PubMed]
  62. Galay, R.L.; Umemiya-Shirafuji, R.; Mochizuki, M.; Fujisaki, K.; Tanaka, T. Iron metabolism in hard ticks (Acari: Ixodidae): The antidote to their toxic diet. Parasitol. Int. 2015, 64, 182–189. [Google Scholar] [CrossRef] [PubMed]
  63. Robinson, M.W.; Hutchinson, A.T.; Dalton, J.P.; Donnelly, S. Peroxiredoxin: A central player in immune modulation. Parasite Immunol. 2010, 32, 305–313. [Google Scholar] [CrossRef] [PubMed]
  64. Hernandez, E.P.; Kusakisako, K.; Talactac, M.R.; Galay, R.L.; Hatta, T.; Fujisaki, K.; Tsuji, N.; Tanaka, T. Glutathione S-transferases play a role in the detoxification of flumethrin and chlorpyrifos in Haemaphysalis longicornis. Parasit. Vectors 2018, 11, 460. [Google Scholar] [CrossRef] [PubMed]
  65. Zhao, M.; Gao, Z.; Ji, X.; Wang, K.; Zhang, S.; Shi, Y.; Song, X.; Yu, Z.; Yang, X. The diverse functions of Mu-class Glutathione S-transferase HrGSTm1 during the development of Hyalomma rufipes with a focus on the detoxification metabolism of cyhalothrin. Parasit. Vectors 2024, 17, 1. [Google Scholar] [CrossRef]
  66. Salinas, A.E.; Wong, M.G. Glutathione S-transferases--a review. Curr. Med. Chem. 1999, 6, 279–309. [Google Scholar] [CrossRef]
  67. Hernandez, E.P.; Talactac, M.R.; Vitor, R.J.S.; Yoshii, K.; Tanaka, T. An Ixodes scapularis glutathione S-transferase plays a role in cell survival and viability during Langat virus infection of a tick cell line. Acta Trop. 2021, 214, 105763. [Google Scholar] [CrossRef]
  68. Citelli, M.; Lara, F.A.; da Silva Vaz, I., Jr.; Oliveira, P.L. Oxidative stress impairs heme detoxification in the midgut of the cattle tick, Rhipicephalus (Boophilus) microplus. Mol. Biochem. Parasitol. 2007, 151, 81–88. [Google Scholar] [CrossRef]
  69. Kumar, D.; Budachetri, K.; Meyers, V.C.; Karim, S. Assessment of tick antioxidant responses to exogenous oxidative stressors and insight into the role of catalase in the reproductive fitness of the Gulf Coast tick, Amblyomma maculatum. Insect Mol. Biol. 2016, 25, 283–294. [Google Scholar] [CrossRef]
  70. Budachetri, K.; Kumar, D.; Karim, S. Catalase is a determinant of the colonization and transovarial transmission of Rickettsia parkeri in the Gulf Coast tick Amblyomma maculatum. Insect Mol. Biol. 2017, 26, 414–419. [Google Scholar] [CrossRef]
  71. Lu, J.; Holmgren, A. The thioredoxin antioxidant system. Free Radic. Biol. Med. 2014, 66, 75–87. [Google Scholar] [CrossRef] [PubMed]
  72. Kusakisako, K.; Galay, R.L.; Umemiya-Shirafuji, R.; Hernandez, E.P.; Maeda, H.; Talactac, M.R.; Tsuji, N.; Mochizuki, M.; Fujisaki, K.; Tanaka, T. 2-Cys peroxiredoxin is required in successful blood-feeding, reproduction, and antioxidant response in the hard tick Haemaphysalis longicornis. Parasit. Vectors 2016, 9, 457. [Google Scholar] [CrossRef]
  73. Kusakisako, K.; Fujisaki, K.; Tanaka, T. The multiple roles of peroxiredoxins in tick blood feeding. Exp. Appl. Acarol. 2018, 75, 269–280. [Google Scholar] [CrossRef]
  74. Kusakisako, K.; Morokuma, H.; Talactac, M.R.; Hernandez, E.P.; Yoshii, K.; Tanaka, T. A Peroxiredoxin From the Haemaphysalis longicornis Tick Affects Langat Virus Replication in a Hamster Cell Line. Front. Cell Infect. Microbiol. 2020, 10, 7. [Google Scholar] [CrossRef]
  75. Hildebrandt, T.; Knuesting, J.; Berndt, C.; Morgan, B.; Scheibe, R. Cytosolic thiol switches regulating basic cellular functions: GAPDH as an information hub? Biol. Chem. 2015, 396, 523–537. [Google Scholar] [CrossRef] [PubMed]
  76. Reddy, K.V.; Yedery, R.D.; Aranha, C. Antimicrobial peptides: Premises and promises. Int. J. Antimicrob. Agents 2004, 24, 536–547. [Google Scholar] [CrossRef]
  77. Zhou, J.; Liao, M.; Ueda, M.; Gong, H.; Xuan, X.; Fujisaki, K. Sequence characterization and expression patterns of two defensin-like antimicrobial peptides from the tick Haemaphysalis longicornis. Peptides 2007, 28, 1304–1310. [Google Scholar] [CrossRef] [PubMed]
  78. Belmonte, R.; Cruz, C.E.; Pires, J.R.; Daffre, S. Purification and characterization of Hb 98-114: A novel hemoglobin-derived antimicrobial peptide from the midgut of Rhipicephalus (Boophilus) microplus. Peptides 2012, 37, 120–127. [Google Scholar] [CrossRef]
  79. Oldiges, D.P.; Parizi, L.F.; Zimmer, K.R.; Lorenzini, D.M.; Seixas, A.; Masuda, A.; da Silva Vaz, I., Jr.; Termignoni, C. A Rhipicephalus (Boophilus) microplus cathepsin with dual peptidase and antimicrobial activity. Int. J. Parasitol. 2012, 42, 635–645. [Google Scholar] [CrossRef]
  80. Nakajima, Y.; Ogihara, K.; Taylor, D.; Yamakawa, M. Antibacterial hemoglobin fragments from the midgut of the soft tick, Ornithodoros moubata (Acari: Argasidae). J. Med. Entomol. 2003, 40, 78–81. [Google Scholar] [CrossRef]
  81. Armstrong, P.B.; Quigley, J.P. Immune function of α2-macrog-lobulin in invertebrates. In Invertebrate Immunology; Rinkevich, B., Müller, W.E.G., Eds.; Springer: Berlin/Heidelberg, Germany, 1996. [Google Scholar]
  82. Buresova, V.; Hajdusek, O.; Franta, Z.; Sojka, D.; Kopacek, P. IrAM-An alpha2-macroglobulin from the hard tick Ixodes ricinus: Characterization and function in phagocytosis of a potential pathogen Chryseobacterium indologenes. Dev. Comp. Immunol. 2009, 33, 489–498. [Google Scholar] [CrossRef] [PubMed]
  83. Zhang, H.; Zhang, W.; Wang, X.; Zhou, Y.; Wang, N.; Zhou, J. Identification of a cysteine-rich antimicrobial peptide from salivary glands of the tick Rhipicephalus haemaphysaloides. Peptides 2011, 32, 441–446. [Google Scholar] [CrossRef] [PubMed]
  84. Zhang, H.; Yang, S.; Gong, H.; Cao, J.; Zhou, Y.; Zhou, J. Functional analysis of a novel cysteine-rich antimicrobial peptide from the salivary glands of the tick Rhipicephalus haemaphysaloides. Parasitol. Res. 2015, 114, 3855–3863. [Google Scholar] [CrossRef] [PubMed]
  85. Silva, F.D.; Rezende, C.A.; Rossi, D.C.; Esteves, E.; Dyszy, F.H.; Schreier, S.; Gueiros-Filho, F.; Campos, C.B.; Pires, J.R.; Daffre, S. Structure and mode of action of microplusin, a copper II-chelating antimicrobial peptide from the cattle tick Rhipicephalus (Boophilus) microplus. J. Biol. Chem. 2009, 284, 34735–34746. [Google Scholar] [CrossRef] [PubMed]
  86. Rodríguez-Camarillo, S.D.; Quiroz-Castañeda, R.E.; Aguilar-Díaz, H.; Vara-Pastrana, J.E.; Pescador-Pérez, D.; Amaro-Estrada, I.; Martínez-Ocampo, F. Immunoinformatic Analysis to Identify Proteins to Be Used as Potential Targets to Control Bovine Anaplasmosis. Int. J. Microbiol. 2020, 2020, 8882031. [Google Scholar] [CrossRef]
  87. Martins, L.A.; Malossi, C.D.; Galletti, M.; Ribeiro, J.M.; Fujita, A.; Esteves, E.; Costa, F.B.; Labruna, M.B.; Daffre, S.; Fogaça, A.C. The Transcriptome of the Salivary Glands of Amblyomma aureolatum Reveals the Antimicrobial Peptide Microplusin as an Important Factor for the Tick Protection Against Rickettsia rickettsii Infection. Front. Physiol. 2019, 10, 529. [Google Scholar] [CrossRef]
  88. Laskowski, M.; Qasim, M.A. What can the structures of enzyme-inhibitor complexes tell us about the structures of enzyme substrate complexes? Biochim. Biophys. Acta 2000, 1477, 324–337. [Google Scholar] [CrossRef]
  89. Chmelař, J.; Kotál, J.; Langhansová, H.; Kotsyfakis, M. Protease Inhibitors in Tick Saliva: The Role of Serpins and Cystatins in Tick-host-Pathogen Interaction. Front. Cell Infect. Microbiol. 2017, 7, 216. [Google Scholar] [CrossRef]
  90. Xu, Z.; Yan, Y.; Zhang, H.; Cao, J.; Zhou, Y.; Xu, Q.; Zhou, J. A serpin from the tick Rhipicephalus haemaphysaloides: Involvement in vitellogenesis. Vet. Parasitol. 2020, 279, 109064. [Google Scholar] [CrossRef]
  91. Xu, Z.; Yan, Y.; Cao, J.; Zhou, Y.; Zhang, H.; Xu, Q.; Zhou, J. A family of serine protease inhibitors (serpins) and its expression profiles in the ovaries of Rhipicephalus haemaphysaloides. Infect. Genet. Evol. 2020, 84, 104346. [Google Scholar] [CrossRef]
  92. Chlastáková, A.; Kaščáková, B.; Kotál, J.; Langhansová, H.; Kotsyfakis, M.; Kutá Smatanová, I.; Tirloni, L.; Chmelař, J. Iripin-1, a new anti-inflammatory tick serpin, inhibits leukocyte recruitment in vivo while altering the levels of chemokines and adhesion molecules. Front. Immunol. 2023, 14, 1116324. [Google Scholar] [CrossRef] [PubMed]
  93. Xu, T.; Lew-Tabor, A.; Rodriguez-Valle, M. Effective inhibition of thrombin by Rhipicephalus microplus serpin-15 (RmS-15) obtained in the yeast Pichia pastoris. Ticks Tick Borne Dis. 2016, 7, 180–187. [Google Scholar] [CrossRef] [PubMed]
  94. Kim, T.K.; Tirloni, L.; Radulovic, Z.; Lewis, L.; Bakshi, M.; Hill, C.; da Silva Vaz, I., Jr.; Logullo, C.; Termignoni, C.; Mulenga, A. Conserved Amblyomma americanum tick Serpin19, an inhibitor of blood clotting factors Xa and XIa, trypsin and plasmin, has anti-haemostatic functions. Int. J. Parasitol. 2015, 45, 613–627. [Google Scholar] [CrossRef] [PubMed]
  95. Kotál, J.; Polderdijk, S.G.I.; Langhansová, H.; Ederová, M.; Martins, L.A.; Beránková, Z.; Chlastáková, A.; Hajdušek, O.; Kotsyfakis, M.; Huntington, J.A.; et al. Ixodes ricinus Salivary Serpin Iripin-8 Inhibits the Intrinsic Pathway of Coagulation and Complement. Int. J. Mol. Sci. 2021, 22, 9480. [Google Scholar] [CrossRef]
  96. Leboulle, G.; Crippa, M.; Decrem, Y.; Mejri, N.; Brossard, M.; Bollen, A.; Godfroid, E. Characterization of a novel salivary immunosuppressive protein from Ixodes ricinus ticks. J. Biol. Chem. 2002, 277, 10083–10089. [Google Scholar] [CrossRef]
  97. Porter, L.; Radulović, Ž.; Kim, T.; Braz, G.R.; Da Silva Vaz, I., Jr.; Mulenga, A. Bioinformatic analyses of male and female Amblyomma americanum tick expressed serine protease inhibitors (serpins). Ticks Tick Borne Dis. 2015, 6, 16–30. [Google Scholar] [CrossRef]
  98. Fredslund, F.; Laursen, N.S.; Roversi, P.; Jenner, L.; Oliveira, C.L.; Pedersen, J.S.; Nunn, M.A.; Lea, S.M.; Discipio, R.; Sottrup-Jensen, L.; et al. Structure of and influence of a tick complement inhibitor on human complement component 5. Nat. Immunol. 2008, 9, 753–760. [Google Scholar] [CrossRef] [PubMed]
  99. Valenzuela, J.G.; Charlab, R.; Mather, T.N.; Ribeiro, J.M. Purification, cloning, and expression of a novel salivary anticomplement protein from the tick, Ixodes scapularis. J. Biol. Chem. 2000, 275, 18717–18723. [Google Scholar] [CrossRef]
  100. Schroeder, H.; Daix, V.; Gillet, L.; Renauld, J.C.; Vanderplasschen, A. The paralogous salivary anti-complement proteins IRAC I and IRAC II encoded by Ixodes ricinus ticks have broad and complementary inhibitory activities against the complement of different host species. Microbes Infect. 2007, 9, 247–250. [Google Scholar] [CrossRef]
  101. Tyson, K.; Elkins, C.; Patterson, H.; Fikrig, E.; de Silva, A. Biochemical and functional characterization of Salp20, an Ixodes scapularis tick salivary protein that inhibits the complement pathway. Insect Mol. Biol. 2007, 16, 469–479. [Google Scholar] [CrossRef]
  102. Nguyen, T.T.; Kim, T.H.; Bencosme-Cuevas, E.; Berry, J.; Gaithuma, A.S.K.; Ansari, M.A.; Kim, T.K.; Tirloni, L.; Radulovic, Z.; Moresco, J.J.; et al. A tick saliva serpin, IxsS17 inhibits host innate immune system proteases and enhances host colonization by Lyme disease agent. PLoS Pathog. 2024, 20, e1012032. [Google Scholar] [CrossRef] [PubMed]
  103. Lu, J.; Wang, K.; Gao, Z.; Zhang, S.; Li, H.; Shi, Y.; Song, X.; Liu, J.; Yu, Z.; Yang, X. Doenitin-1: A novel Kunitz family protein with versatile functions during feeding and reproduction of the tick Haemaphysalis doenitzi. Front. Vet. Sci. 2022, 9, 872244. [Google Scholar] [CrossRef] [PubMed]
  104. Assumpção, T.C.; Ma, D.; Mizurini, D.M.; Kini, R.M.; Ribeiro, J.M.; Kotsyfakis, M.; Monteiro, R.Q.; Francischetti, I.M. In Vitro Mode of Action and Anti-thrombotic Activity of Boophilin, a Multifunctional Kunitz Protease Inhibitor from the Midgut of a Tick Vector of Babesiosis, Rhipicephalus microplus. PLoS Negl. Trop. Dis. 2016, 10, e0004298. [Google Scholar] [CrossRef] [PubMed]
  105. Soares, T.S.; Watanabe, R.M.; Tanaka-Azevedo, A.M.; Torquato, R.J.; Lu, S.; Figueiredo, A.C.; Pereira, P.J.; Tanaka, A.S. Expression and functional characterization of boophilin, a thrombin inhibitor from Rhipicephalus (Boophilus) microplus midgut. Vet. Parasitol. 2012, 187, 521–528. [Google Scholar] [CrossRef]
  106. Liao, M.; Zhou, J.; Gong, H.; Boldbaatar, D.; Shirafuji, R.; Battur, B.; Nishikawa, Y.; Fujisaki, K. Hemalin, a thrombin inhibitor isolated from a midgut cDNA library from the hard tick Haemaphysalis longicornis. J. Insect Physiol. 2009, 55, 164–173. [Google Scholar] [CrossRef]
  107. Lovato, D.V.; Nicolau de Campos, I.T.; Amino, R.; Tanaka, A.S. The full-length cDNA of anticoagulant protein infestin revealed a novel releasable Kazal domain, a neutrophil elastase inhibitor lacking anticoagulant activity. Biochimie 2006, 88, 673–681. [Google Scholar] [CrossRef]
  108. de Marco, R.; Lovato, D.V.; Torquato, R.J.; Clara, R.O.; Buarque, D.S.; Tanaka, A.S. The first pacifastin elastase inhibitor characterized from a blood sucking animal. Peptides 2010, 31, 1280–1286. [Google Scholar] [CrossRef]
  109. Kellenberger, C.; Roussel, A. Structure-activity relationship within the serine protease inhibitors of the pacifastin family. Protein Pept. Lett. 2005, 12, 409–414. [Google Scholar] [CrossRef]
  110. Villar, M.; Ayllón, N.; Busby, A.T.; Galindo, R.C.; Blouin, E.F.; Kocan, K.M.; Bonzón-Kulichenko, E.; Zivkovic, Z.; Almazán, C.; Torina, A.; et al. Expression of Heat Shock and Other Stress Response Proteins in Ticks and Cultured Tick Cells in Response to Anaplasma spp. Infection and Heat Shock. Int. J. Proteomics 2010, 2010, 657261. [Google Scholar] [CrossRef]
  111. Vos, M.J.; Carra, S.; Kanon, B.; Bosveld, F.; Klauke, K.; Sibon, O.C.; Kampinga, H.H. Specific protein homeostatic functions of small heat-shock proteins increase lifespan. Aging Cell 2016, 15, 217–226. [Google Scholar] [CrossRef]
  112. Will, T.; Schmidtberg, H.; Skaljac, M.; Vilcinskas, A. Heat shock protein 83 plays pleiotropic roles in embryogenesis, longevity, and fecundity of the pea aphid Acyrthosiphon pisum. Dev. Genes. Evol. 2017, 227, 1–9. [Google Scholar] [CrossRef] [PubMed]
  113. Morrow, G.; Battistini, S.; Zhang, P.; Tanguay, R.M. Decreased lifespan in the absence of expression of the mitochondrial small heat shock protein Hsp22 in Drosophila. J. Biol. Chem. 2004, 279, 43382–43385. [Google Scholar] [CrossRef] [PubMed]
  114. Liu, L.; He, X.M.; Feng, L.L.; Duan, D.Y.; Zhan, Y.; Cheng, T.Y. Cloning of four HSPA multigene family members in Haemaphysalis flava ticks. Med. Vet. Entomol. 2020, 34, 192–200. [Google Scholar] [CrossRef]
  115. Vora, A.; Taank, V.; Dutta, S.M.; Anderson, J.F.; Fish, D.; Sonenshine, D.E.; Catravas, J.D.; Sultana, H.; Neelakanta, G. Ticks elicit variable fibrinogenolytic activities upon feeding on hosts with different immune backgrounds. Sci. Rep. 2017, 7, 44593. [Google Scholar] [CrossRef] [PubMed]
  116. He, X.M.; Liu, L.; Cheng, T.Y. HSC70 from Haemaphysalis flava (Acari: Ixodidae) exerts anticoagulation activity in vitro. Ticks Tick Borne Dis. 2019, 10, 170–175. [Google Scholar] [CrossRef] [PubMed]
  117. Liu, Y.-K.; Liu, G.-H.; Liu, L.; Wang, A.-B.; Cheng, T.-Y.; Duan, D.-Y. Comparative analysis of the anticoagulant activities and immunogenicity of HSC70 and HSC70(TKD) of Haemaphysalis flava. Parasit. Vectors 2022, 15, 411. [Google Scholar] [CrossRef]
  118. Friesen, K.J.; Dixon, M.; Lysyk, T.J. Embryo Development and Morphology of the Rocky Mountain Wood Tick (Acari: Ixodidae). J. Med. Entomol. 2016, 53, 279–289. [Google Scholar] [CrossRef]
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Figure 1. SDS-PAGE analysis of protein extract from H. hystricis eggs. A total of 80 μg of protein extract was loaded per sample. Lane M represents the 15–250 kDa molecular weight marker. D1, D7, D14, and D21 correspond to eggs incubated for 1, 7, 14, and 21 days, respectively. Bands 1–12 indicate the protein bands excised for further analysis.
Figure 1. SDS-PAGE analysis of protein extract from H. hystricis eggs. A total of 80 μg of protein extract was loaded per sample. Lane M represents the 15–250 kDa molecular weight marker. D1, D7, D14, and D21 correspond to eggs incubated for 1, 7, 14, and 21 days, respectively. Bands 1–12 indicate the protein bands excised for further analysis.
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Figure 2. Top 4 most prominent proteins identified in 12 bands from day-one eggs.
Figure 2. Top 4 most prominent proteins identified in 12 bands from day-one eggs.
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Figure 3. Analysis of protein dynamics in H. hystricis tick eggs across different incubation days. Each sample was spiked with the stable isotope iRT KIT peptide as an internal standard. Tryptic peptides were analyzed using the nLC-1200 system. Protein abundances at 7, 14, and 21 days of incubation were normalized to the levels observed at day. D1, D7, D14, and D21 correspond to eggs incubated for 1, 7, 14, and 21 days, respectively.
Figure 3. Analysis of protein dynamics in H. hystricis tick eggs across different incubation days. Each sample was spiked with the stable isotope iRT KIT peptide as an internal standard. Tryptic peptides were analyzed using the nLC-1200 system. Protein abundances at 7, 14, and 21 days of incubation were normalized to the levels observed at day. D1, D7, D14, and D21 correspond to eggs incubated for 1, 7, 14, and 21 days, respectively.
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Table 1. High abundance transtripts (iBAQ values exceeding 1.00 × 1010) identified in eggs of H. hystricis on day-one incubation.
Table 1. High abundance transtripts (iBAQ values exceeding 1.00 × 1010) identified in eggs of H. hystricis on day-one incubation.
NoTranscripts ID in PRJNA1168713Alignment Entry and OverviewE ValueScoreIdentity (%)iBAQ
TRANSPORTERS Cluster-17602.43418A0A8X8MG03, Vitellin-a, Haemaphysalis flava09.17 × 10393.30%1.51 × 1010
Cluster-17602.25911A0A411G179, Vitellogenin, Haemaphysalis flava08.9 × 10387.50%1.39 × 1010
ENZYMESCluster-17602.15229A0A6M2CKA2, Yolk cathepsin, Rhipicephalus microplus01.37 × 10366.80%7.67 × 109
Cluster-11877.0A0A8K1PH81, Heme-binding asparlic proteinase, Rhipicephalus microplus01.35 × 10367.50%1.40 × 109
IMMUNITY-RELATED PROTEINCluster-17602.29432A0A5B9BYB0, Cysteine-rich protein, Haemaphysalis flava3.10 × 101108.10 × 10286.70%1.11 × 109
Cluster-17602.8619A0A6M2D6P5, His-rich 1 fat body overexpressed, Rhipicephalus microplus5.10 × 10171.88 × 10238.10%1.50 × 109
Cluster-17602.7590A0A6M2D6P5, His-rich 1 fat body overexpressed, Rhipicephalus microplus1.80 × 10171.91 × 10240.20%2.16 × 109
PROTEANASE INHIBITORSCluster-17602.31952A0A5P8H6S1, Serpin-a, Haemaphysalis longicornis7.40 × 101581.17 × 10361.20%1.18 × 109
Cluster-11893.0A0A034WTW4, Serine proteinase inhibitor, Rhipicephalus microplus1.10 × 10292.69 × 10256.80%4.20 × 109
Cluster-17602.30986A0A034WTW0, Kunitz domain-containing protein 1, Rhipicephalus microplus01.83 × 10334.50%4.49 × 109
Cluster-17602.10962A0A8F1NJE0, Neutrophil elastase inhibitor, Haemaphysalis flava5.70 × 10302.70 × 10253.50%1.63 × 109
Cluster-17602.18555A0A8F1NJE0, Neutrophil elastase inhibitor, Haemaphysalis flava7.40 × 10493.94 × 10273.50%1.25 × 1010
Cluster-17602.35730A0A8F1NJE0, Neutrophil elastase inhibitor, Haemaphysalis flava6.40 × 10473.92 × 10272.50%1.15 × 1010
OTHERSCluster-17805.0B7QGW1, Secreted protein, Ixodes scapularis2.40 × 10192.07 × 10242.30%1.97 × 109
Cluster-12057.0Q202J4, Dermonecrotic toxin SPH, Ixodes scapularis9.60 × 10361.94 × 10244.90%1.44 × 109
Cluster-17602.12808A0A182TMR5, Cuticular protein, Anopheles melas2.00 × 10153.33 × 10229.70%2.08 × 109
iBAQ: intensity-based absolute quantification.
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Tang, Q.; Cheng, T.; Liu, W. Egg Protein Compositions over Embryonic Development in Haemaphysalis hystricis Ticks. Animals 2024, 14, 3466. https://doi.org/10.3390/ani14233466

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Tang Q, Cheng T, Liu W. Egg Protein Compositions over Embryonic Development in Haemaphysalis hystricis Ticks. Animals. 2024; 14(23):3466. https://doi.org/10.3390/ani14233466

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Tang, Qiwu, Tianyin Cheng, and Wei Liu. 2024. "Egg Protein Compositions over Embryonic Development in Haemaphysalis hystricis Ticks" Animals 14, no. 23: 3466. https://doi.org/10.3390/ani14233466

APA Style

Tang, Q., Cheng, T., & Liu, W. (2024). Egg Protein Compositions over Embryonic Development in Haemaphysalis hystricis Ticks. Animals, 14(23), 3466. https://doi.org/10.3390/ani14233466

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