Developmental Proteomics Reveals the Dynamic Expression Prole of Global Proteins of Haemaphysalis Longicornis (Parthenogenesis)

Background: Ticks are important parasites that cause more diseases than most other animal parasites. Haemaphysalis longicornis is used as an experimental animal model for the study of three-host ticks due to its special life cycle and easy maintenance in the laboratory and in its reproduction. The life cycle of H. longicornis goes through a tightly regulated life cycle to adapt to the changing host and environment, and these stages of transition are also accompanied by proteome changes in the body. Methods: In this study, the aim was to use the isobaric tags for relative and absolute quantication (iTRAQ) technique to systematically describe and analyze the dynamic expression of protein and the molecular basis of the proteome of H. longicornis in seven differential developmental stages (eggs, unfed larvae, fed larvae, unfed nymphs, fed nymphs unfed adults, and fed adults). Results: A total of 2,059 proteins were identied, and their expression proles were classied at different developmental stages. In addition, it was found that tissue and organ development-related proteins and metabolism-related proteins showed that they were involved in different physiological processes throughout the life cycle through the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of the differentially expressed proteins (DEPs). More importantly, we found that the upregulated proteins of fed adult ticks were mainly related to yolk absorption, degradation, and ovarian development-related proteins. The abundance of the cuticle proteins in the unfed stages were signicantly higher compared with those of the fed ticks in the previous stages. Conclusions: In short, the protein spectrum changes identied in this study provide a reference proteome for future studies of tick functional proteins and provide candidate targets for elucidating tick development and developing new tick control strategies. B phase B mobile phase B component peak centrifuged nanoliter column B linearly 43–48 phase 48–50 phase B from mobile phase B to the mobile phase B, 55-65 and 5%, phase B. end nanoliter liquid phase structural consistent of cuticle, and protein kinase activity; and in the biological process: microtubule-based process, homeostatic process, protein-DNA complex assembly, and cellular component organization.


Introduction
Ticks are globally-distributed, blood-sucking ectoparasites that are related to tick-borne diseases. The impact of tick-borne diseases is staggering, especially in developing countries, where tick-borne diseases affect about 80% of the world's cattle, with economic losses estimated to be between US $13.9 billion and US $18.7 billion [1,2].
The peptides separated by liquid phase were ionized by a nanoESI source, and then entered into a tandem mass spectrometer Q-Exactive HF (Thermo Fisher Scienti c, San Jose, CA) for data-dependent acquisition (DDA) mode detection. The main parameters were as follows: the ion source voltage was set to 1.6 kV; the scanning range of the rst-stage mass spectrometry was 350~1,600 m/z; the resolution was set to 60,000 m; the initial m-stroke z of the second-stage mass spectrometry was xed to 100; and the resolution was 15,000. The screening conditions for the parent ions of the secondary fragmentation were as follows: the charge was 2+ to 6+, and the parent ions with peak intensity of more than 10,000 were in the top 30. The ion fragmentation mode was higher-energy C-trap dissociation (HCD), and fragment ions were detected in Orbitrap. The dynamic exclusion time was set to 30 s, and the automatic gain control (AGC) was set to rst-level 3E6 and second-level 1E5.

Database search and bioinformatics analysis
The original MS data was converted into a general format (.mgf) le with Proteome Discoverer 1.4, and then the data le was used to query the tick-related database. ProteinPilot protein software 4.5 (AB SCIEX) was used for further identi cation and quanti cation of proteins. In order to lter the results, we used an error detection rate of less than 0.01 for the identi cation, and for the quanti cation, a con dence level of 95% or an unused con dence score greater than 1.3a was used. For the DEPs, values | log 2 -fold change | > 1 were regarded as upregulated or down-regulated proteins, respectively.
Functional classi cation of the DEPs was carried out according to GO annotation and http://www.geneontology.org analysis. The DEPs were divided into three categories, namely, molecular function, biological processes, and cellular components. The KEGG (http://www.kegg.jp/kegg/) was used to predict the molecular functions, biological processes, and important DEP pathways.

Quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis
Six proteins (CRK, otillin, Mo-25, dystrophin, septin-1, and septin-2) were randomly selected from the protein library built by iTRAQ, and these genes were compared at the transcriptional level by RT-qPCR. The rst cDNA strand was synthesized using the total RNA from the samples from seven stages (egg, unfed larva, fed larva, unfed nymph, unfed adult, and unfed adult) using the PrimeScript Reverse Transcriptase kit (TaKaRa, Dalian, China), according to the manufacturer's instructions. The RT-qPCR was carried out in accordance with the methods described earlier and relative transcript levels were calculated using the 2 ΔΔCt method [13]. The information on the primers of the selected gene and the reference gene beta-actin is shown in Table 1.

Statistical Analysis
All statistical data were processed with GraphPad Prism software version 7 (GraphPad, CA, USA), and the data were analyzed by Student's ttest. P < 0.05 was considered statistically signi cant.

Results And Discussion
The development of H. longicornis is accompanied by many changes in the host, such as sucking blood, molting, and spawning. In the whole process, the occurrence, development, and degeneration of tissues and organs are also accompanied by changes in the protein pro le. The lack of detailed and comprehensive protein dynamic spectrum during the development of H. longicornis limits the research of development of H. longicornis, the screening of drug resistance genes, and the development of anti-tick drugs and other related studies. The iTRAQ quantitative proteomics is a powerful and novel tool for characterizing protein changes among different samples, which has been well veri ed in a variety of organisms, and helps people to analyze the protein pro les of many organisms under different conditions and different developmental stages [14][15][16]. Therefore, in this experiment, in order to clearly understand the differences in the physiological functions of H. longicornis at different developmental stages, especially, in order to identify, quantify, and compare the protein expression pro les of H. longicornis at different developmental stages, we used iTRAQ quantitative proteomics technology to analyze the related functions of H. longicornis proteins at different developmental stages.
Overview of main data and protein identi cation In this study, iTRAQ was used to identify the proteomic at different stages of the life cycle of H. longicornis; that is, egg, unfed larvae, fed larvae, unfed nymph, fed nymph, unfed adult, and fed adult. In the three repeated experiments, a total of 2,056 proteins were identi ed from 4,405 peptides, which were matched with 2,608,862 spectra at a false discovery rate of 1% ( Table 2). As shown in Figure 1A, the number of proteins identi ed in the three repeated experiments was 1,325; 1,333; and 1,504, respectively, while the 812 proteins were identi ed to be shared in the three repeats. Most of the proteins were identi ed by one peptide; speci cally, 939.7 ± 45.28 (45.71%) proteins were identi ed based on one peptide. More than 218.7 ± 8.74 (10.64%) proteins were identi ed based on two peptides, 87 ± 4.35 (4.25%) proteins were identi ed based on three peptides, and about 810.5 ± 24.35 (39.45%)proteins were identi ed by more than three peptides ( Figure 1B). The sequence coverage of a speci cally identi ed protein is estimated as the percentage of matching amino acids between the identi ed peptides with more than 95% con dence divided by the total number of amino acids in the protein sequence. The sequence coverage of 473.7 ± 38.35 (37.47%) proteins was less than 0-10%, while the sequence coverage of 393 ± 15.57 (23.15%) proteins was 10-20%.

Protein quanti cation
The H. longicornis tick has a complicated life cycle, and the molecular basis of its growth and development is still poorly understood. In order to study the protein pro le during its growth process, we have obtained samples from seven differential development stages (egg, unfed larvae, fed larvae, unfed nymph, fed nymph, unfed adult, and fed adult). A proteomic analysis was performed, and the proteins with |log 2 -fold change|> 1 and P < 0.05 were considered DEPs. A pairwise comparison of the proteins of each successive stage was conducted, and it was found that compared with those of eggs, the unfed larvae had 124 up-regulated proteins and 83 down-regulated proteins; compared with those of hungry young ticks, there were 94 up-regulated proteins and 79 down-regulated proteins in the fed larvae; compared with the those of fed larvae, the unfed nymph had 88 up-regulated proteins and 89 down-regulated proteins; compared with those of unfed nymph, there were 86 up-regulated proteins in the ticks and 101 down-regulated proteins in the fed nymph; compared with those of fed nymph, there were 99 up-regulated proteins and 88 down-regulated proteins in the unfed adult; and compared with those of unfed adult, there were 70 upregulated proteins and 87 down-regulated proteins in the fed adult. Figure 2 shows the number of DEP at different developmental stages.

RT-qPCR analysis
The transcriptional levels of six genes (CRK, otillin, Mo-25, dystrophin, septin-1, and septin-2) were examined at seven stages. Through the analysis of the results, we found that the change of their transcriptional levels was not consistent with the trend of protein levels in iTRAQ at different developmental stages (Table 3 and Fig. 3). The reason for this result is mainly attributed to the fact that RNA level is only a moderate proxy for protein abundance and does not fully represent protein expression abundance. These results highlight that it is necessary to analyze the differentiation mechanism components of H. longicornis at the protein level, which are involved in basic biological processes such as signal transduction, substance transport, catalytic activity, metabolism, and so on.
Expression pro le of the identi ed proteins

Chitin-binding proteins
In this experiment, three chitin binding proteins (Cluster-30738.173199, Cluster-30738.190566, and Cluster-30738.187492) were identi ed. Among them, the two peritrophic membrane chitin-binding proteins shared the same expression trend; that is, they were decreased in the process of egg hatching into unfed larva, and then increased signi cantly with blood sucking. On the other hand, in the process of entering the next stage after blood sucking, it showed a signi cant downward trend, and then reached a peak in the fed adult (Table 4). The peritrophic membrane (PM) is an important organ of blood-sucking arthropods, which provides protection for the microvilli of digestive tract epithelial cells and as a sturdy barrier to protect the intestinal tract from physical damage caused by the structure of food intake and the invasion of parasites and other pathogens [17]. Moreover, many studies have used histochemical and biochemical techniques to show the presence of chitin on the perineal membrane [18,19]. A previous study found that the PM of H. longicornis was signi cantly different between the larvae and the adult stages, and the presence of PM chitin-binding proteins was observed [20]. Similarity, in this study, we identi ed two kinds of PM chitin-binding proteins, both of which showed the same upward trend in the process of blood intake, which is consistent with previous studies, and can also explain their protective role in the process of blood uptake.

Digestion-related proteins
The digestion of blood provides energy and nutrients for maintaining the growth and metabolism of ticks, which is a complex process, requiring the cooperation of a variety of proteins to process and deal with the hemoglobin ingested, and then into their own nutrients [21]. In this study, a variety of proteins related to digestion were found, which would help to use dynamic strategies to explain and clarify the blood digestion process during the development of H. longicornis.
The differences in the expression of proteins related to digestion were analyzed in the different stages in the experiment. Interestingly, we found four trypsin proteins (Cluster-30738.179855, Cluster-30738.158249, Cluster-30738.86970, and Cluster-30738.127284). They increased signi cantly from unfed nymph to fed nymph as well as in unfed adult to fed adult. In addition, we also found three carboxypeptidases proteins (Cluster-30738.164810, Cluster-30738.108012, and Cluster-30738.136271). The expression of Cluster-30738.164810 showed low abundance in both eggs and larva stages, and then increased rapidly from unfed nymph and lasted until fed adult. In addition, the expression abundance of two proteins (Cluster-30738.108012 and Cluster-30738.136271) increased from eggs to the larva stage. After that, they showed upward trends from unfed stages to corresponding fed stages. Leucine aminopeptidase (Cluster-30738.169581) increased from eggs to the unfed larva stage, then began to decrease gradually and decreased to the lowest level until unfed adults (Table 5).

Vitellogenin proteins
During the development of ticks, vitellogenin (Vg) is synthesized as a high-molecular weight precursor in body fat, gut, and ovary. After that, the Vg is released into the hemolymph and absorbed and accumulated by oocytes through receptor-mediated endocytosis. At this time, it is named Vt, which is an important source of nutrients for embryonic development [22,23]. In this study, six vitellogenin proteins were identi ed: Vg1, Vg2, Vg3, Vg4, Vg5, and Vg6 (Cluster-30738.183992, Cluster-30738.173105, Cluster-30738.197239, Cluster-30738.175424, Cluster-30738.173278, and Cluster-30738.195117). Among them, Vg2, Vg3, and Vg6 showed the same expression pattern. The expression abundance of these Vg proteins increased signi cantly from egg hatching to unfed larva but began to decrease during the development of unfed larva to fed larva and increased again during molting into unfed nymph. Then, after sucking blood to the fed nymph stage, the content decreased again. While molting into the unfed adult stage, their content increased again, and then declined again after the last bloodsucking into the engorged adult stage. However, the expression abundance of Vg1 increased signi cantly from unfed nymph to fed nymph and from unfed adults to fed adults. The results of the Vg4 and Vg5 showed that the expression abundance increased signi cantly from unfed larva to fed larva and from unfed adults to fed adults (Table 6). As early as 2010, scientists successfully annotated Vg1, Vg2, and Vg3 in H. longicornis and identi ed the protein size of these three Vgs. Also, they observed a rapid increase in Vg2 and Vg3 transcription levels in the body fat on the second day in feeding, a signi cant increase in Vg1 transcription in the midgut on the fourth day, and an increase in the mRNA expression of Vg2 in the ovary from the fourth day in feeding.
To explore their role in the development of H. longicornis, through RNAi technology, it was found that the knockdown of Vgs could signi cantly affect the full blood weight of ticks in eld teaching, and Vgs are necessary for egg weight and oviposition [23].
In 2018, scientists explored the ovariogenesis of Boophilus microplus and identi ed seven Vt peptides, which are the corresponding products of ve different Vgs (Vg1, Vg2, Vg3, Vg4, and Vg5). They were observed to increase during the feeding phase, and most of which increased rapidly at the end of blood feeding [24].
In this study, it was found that the six Vgs showed different expression patterns in the different developmental stages of H. longicornis, suggesting that they may play different roles in different tissues and physiological processes, which needs to be further explored in the future.

Cuticle proteins
The cuticle of ticks is an important defense tissue, which can resist bad weather and other physical injuries, H. longicornis need undergo two molts in its lifetime, during which a lot of cuticle-related proteins undergo change [25]. In the blood-sucking process of ticks, the cuticle protein begins to increase, while in the molting process, the old epidermal protein will be absorbed, and the content will decrease; at the same time, it will gradually synthesize new cuticle proteins until the end of molting. In Liu's paper, the cuticle protein CPR1 is involved in the molting process of H. longicornis and is regulated by miRNA [13].
Thirteen cuticle proteins were found in this study. From the comparison of seven different developmental stages, the expression of these cuticle proteins showed two expression patterns: the rst pattern,  In this study, cuticle-associated proteins showed different expression patterns-one part showed an upward trend in the satiety stage, and the other showed a downward trend. This opposite expression trends implies that there may be great differences in the structure and function of the cuticle proteins, which need to be further analyzed in terms of their protein structure, family classi cation, and related functional studies.

Membrane proteins
Biological process is a circular network, and membrane protein is an important hub in the network, which plays an important physiological role in organisms, such as cell proliferation and differentiation, energy conversion, signal transduction, and material transport. In addition, most drugs also achieve a therapeutic effect by interacting with membrane proteins [26].
A total of 12 membrane proteins were found in this study, which were divided into three patterns by the expression patterns in different developmental stages in the H. longicornis: the expression of the rst class, Cluster-30738.172187, was relatively steady at different developmental stages, and there was no obvious stage speci city.

Salivary proteins
Salivary gland is an important osmoregulation organ of ticks. Whether for a long time away from the host or during the feeding period of the host, the salivary glands are essential for maintaining the growth, development, and metabolism of ticks [27]. Furthermore, salivary glands and saliva play key roles in the transmission of pathogenic microorganisms to the host [28]. By using the psiblast tool, scientists built the TickSialoFam (TSF) database, a publishable database that can help annotate tick sialo transcriptomes [29].
Under the stimulation of blood sucking, the salivary glands will develop and enlarge rapidly, and this process will also be accompanied by changes in a large number of salivary gland-related proteins. A total of ve salivary gland-associated proteins were identi ed in this experiment, and they were classi ed into two classes according to their expression patterns in seven different developmental stages. The expression abundance of the rst class, Cluster-30738.172529, Cluster-30738.173721, and Cluster-30738.175111, increased rapidly in the process of blood sucking, and the expression of these proteins would continue to increase with the process of development in H. longicornis. The expression abundance of the second class, Cluster-30738.164072, increased rapidly in the early stage of development (eggs hatched into unfed larva) and increased signi cantly during the development from unfed nymphs to fed adult (Table 9).

Secreted proteins
Secreted proteins (SP) present in parasites contribute directly or indirectly to the survival of parasites. In addition, parasites need to adapt to different hosts as well as to physiological changes during development, and SP proteins play an important role [30] in these processes.
In this experiment, a total of 37 secreted proteins were identi ed, and many proteins also showed a strong regularity and speci c upregulated expression at different developmental stages. These secreted proteins were mainly divided into three classes by collating the data.  (Table 10). Compared with eggs and unfed larvae ticks, the results of the GO analysis showed the following: for the molecular function: catalytic activity, binding, transport activity, structural molecular activity, and molecular function regulation; for the cell composition: cells, cell components, organs, membrane components, and organ components; and for the biological process: cellular process, metabolic process, regulation of biological functions, stimulus response, and recognition of cell composition/biological inheritance.
Similarly, we also found in the results that compared with the starvation phase, the GO analysis results in the fed stage were as follows: in the cell composition: cell membrane, ribosomes, RNA-induced silencing complex (RSIC), RNAi effector complex; in the molecular function: synthesis, enzyme activity, inhibitor enzyme activity, peptidase activity, synthetase activity, and epidermal composition; and in the biological processes: organic substance biosynthetic, organic substance catabolic, cofactor metabolic, cellular biosynthetic, and carbohydrate derivative metabolic.
In the unfed phase, compared with the previous fed phase, the enrichment results of the GO entries were as follows: in the cell composition: DNA packaging complex, protein-DNA complex, nucleosome, and chromatin; in the molecular function: lipid transporter activity, transporter activity, protein heterodimerization activity, structural consistent of cuticle, and protein kinase activity; and in the biological process: microtubule-based process, homeostatic process, protein-DNA complex assembly, and cellular component organization.

KEGG analysis of the DEPs
In order to further determine the biological pathways in which these differential proteins are involved in the development of H.  Figures 6 and 7).
Among these pathways, it was found that compared with the unfed stage, the signal pathways enriched by the up-regulated proteins in the fed stage mainly included the digestive system, immune system, endocrine system, environmental adaptation, and infectious diseases (viral, and bacterial), signal transduction, cellular community-eukaryotes, cell growth and death, and transport and catabolism.
In the unfed stage, compared with the previous fed stage, the main enriched signal pathways included the excretory system, nervous system, aging, development, cardiovascular diseases, folding, sorting and degradation, replication and repair, and cell motility.

Conclusions
This is the rst in-depth overview of the protein spectrum of H. longicornis (parthenogenesis), which could be of great signi cance for revealing the molecular architecture of ticks with complex life cycle. Our data provides strong molecular support for the use of H. longicornis as a powerful model for studying tick development and reveal a group of proteins. These proteomes have expanded to play a key role in biological regulation such as digestion, molting, ovarian development and immunomodulation. Overall, this is a report on the overall proteomics of H. longicornis, which will help us to understand the complex process of tick development, and the membrane-associated proteins and secretory proteins described in this paper will also help to nd new target proteins and provide a theoretical basis and candidates for improving tick control strategies.

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