Overview of main data and protein identification
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 identified 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 identified in the three repeated experiments was 1,325; 1,333; and 1,504, respectively, while the 812 proteins were identified to be shared in the three repeats. Most of the proteins were identified by one peptide; specifically, 939.7 ± 45.28 (45.71%) proteins were identified based on one peptide. More than 218.7 ± 8.74 (10.64%) proteins were identified based on two peptides, 87 ± 4.35 (4.25%) proteins were identified based on three peptides, and about 810.5 ± 24.35 (39.45%)proteins were identified by more than three peptides (Figure 1B).
Table 2
Spectra, peptide, and protein identified by the isobaric tags for relative and absolute quantification (iTRAQ)
| Total-spectra | Spectra | Unique-spetra | Peptide | Unique-peptide | Protein-identified |
Repeat 1 | 839,950 | 7,932 | 7,109 | 2,769 | 2,663 | 1,325 |
Repeat 2 | 829,948 | 8,019 | 7,163 | 2,781 | 2,673 | 1,333 |
Repeat 3 | 938,964 | 8,951 | 7,961 | 2,939 | 2,844 | 1,504 |
Total | 2,608,862 | 24,902 | 22,233 | 4,405 | 4,295 | 2,056 |
The sequence coverage of a specifically identified protein is estimated as the percentage of matching amino acids between the identified peptides with more than 95% confidence 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%. Moreover, 825 ± 15.57 (40.15%) proteins were determined to have a sequence coverage of more than 40%. The MS data has been deposited in iProX (Integrated Proteome Resources, http://www.iprox.org/) under the main accession number OMIX707 (Figure 1C).
Expression profile of the identified proteins
Chitin-binding proteins
In this experiment, three chitin binding proteins (Cluster-30738.173199, Cluster-30738.190566, and Cluster-30738.187492) were identified. 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 significantly with blood sucking. On the other hand, in the process of entering the next stage after blood sucking, it showed a significant downward trend, and then reached a peak in the fed adult (Table 4).
Table 4
Protein | ID | Mean_Ratio HLUL-VS-HLEE | Mean_Ratio HLFL-VS-HLUL | Mean_Ratio HLUN-VS-HLFL | Mean_Ratio HLFN-VS-HLUN | Mean_Ratio HLUA-VS-HLFN | Mean_Ratio HLFA-VS-HLUA |
Peritrophic membrane chitin-binding protein, putative [Ixodes scapularis] | Cluster-30738.190566 | 0.58 | 2.69 | 0.52 | 2.07 | 0.3 | 3.4 |
Chitin-binding peritrophin-A, putative [Ixodes scapularis] | Cluster-30738.187492 | 1.13 | 0.43 | 1.2 | 0.8 | 0.66 | 1.62 |
Peritrophic membrane chitin-binding protein, putative [Ixodes scapularis] | Cluster-30738.77125 | 1.19 | 1.71 | 0.65 | 2.85 | 0.23 | 2.41 |
*HLEE, egg; HLUL, unfed larva; HLFL, fed larva; HLUN, unfed nymph; HLFN, fed nymph; HLUA, unfed adult; HLFA, fed adult. |
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 significantly different between the larvae and the adult stages, and the presence of PM chitin-binding proteins was observed [20]. Similarity, in this study, we identified 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 significantly 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).
Table 5
Digestion-related proteins
Protein | ID | Mean_Ratio HLUL-VS-HLEE | Mean_Ratio HLFL-VS-HLUL | Mean_Ratio HLUN-VS-HLFL | Mean_Ratio HLFN-VS-HLUN | Mean_Ratio HLUA-VS-HLFN | Mean_Ratio HLFA-VS-HLUA |
Trypsin | Cluster-30738.179855 | 1.2 | 1.51 | 0.9 | 1.03 | 1.02 | 1.14 |
Trypsin-3 | Cluster-30738.158249 | 1.21 | 0.84 | 0.62 | 1.69 | 0.78 | 1.54 |
trypsin-1 | Cluster-30738.86970 | 0.51 | 0 | 0.43 | 4.41 | 0.48 | 2.03 |
Trypsin2 | Cluster-30738.127284 | 0.77 | 0 | 0.63 | 1.37 | 0.52 | 2.58 |
Carboxypeptidas | Cluster-30738.164810 | 0.38 | 0.1 | 3.95 | 0.45 | 2.2 | 2.44 |
Serine carboxypeptidase | Cluster-30738.108012 | 1.14 | 1.76 | 0.75 | 1.22 | 0.93 | 1.15 |
Serine carboxypeptidase 2 | Cluster-30738.136271 | 1.19 | 1.61 | 0.81 | 1.26 | 1.14 | 0.66 |
Leucine aminopeptidase | Cluster-30738.169581 | 1.49 | 1.28 | 1.25 | 0.96 | 0.94 | 0.66 |
*HLEE, egg; HLUL, unfed larva; HLFL, fed larva; HLUN, unfed nymph; HLFN, fed nymph; HLUA, unfed adult; HLFA, fed adult. |
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 identified: 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 significantly 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 significantly 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 significantly from unfed larva to fed larva and from unfed adults to fed adults (Table 6).
Table 6
Vitellogenin (Vg)-related proteins
Protein | ID | Mean_Ratio HLUL-VS-HLEE | Mean_Ratio HLFL-VS-HLUL | Mean_Ratio HLUN-VS-HLFL | Mean_Ratio HLFN-VS-HLUN | Mean_Ratio HLUA-VS-HLFN | Mean_Ratio HLFA-VS-HLUA |
Vitellogenin-1 | Cluster-30738.173246 | 0.52 | 0.44 | 0.66 | 1.46 | 0.66 | 1.99 |
Vitellogenin-2 | Cluster-30738.151517 | 1.57 | 0.73 | 2.93 | 0.39 | 2.32 | 0.44 |
Vitellogenin-3 | Cluster-30738.197239 | 1.25 | 0.87 | 1.53 | 0.67 | 1.3 | 0.98 |
Vitellogenin-4 | | / | 1.57 | 1.06 | 0.92 | 1.3 | 1.14 |
Vitellogenin-5 | | / | 1.95 | 1.66 | 0.73 | 0.96 | 2.01 |
Vitellogenin-6 | Cluster-30738.195117 | 1.43 | 0.97 | 1.7 | 0.66 | 1.47 | 0.51 |
*HLEE, egg; HLUL, unfed larva; HLFL, fed larva; HLUN, unfed nymph; HLFN, fed nymph; HLUA, unfed adult; HLFA, fed adult. |
As early as 2010, scientists successfully annotated Vg1, Vg2, and Vg3 in H. longicornis and identified 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 significant 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 significantly affect the full blood weight of ticks in field teaching, and Vgs are necessary for egg weight and oviposition [23].
In 2018, scientists explored the ovariogenesis of Boophilus microplus and identified seven Vt peptides, which are the corresponding products of five 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 first pattern, Cluster-30738.125201, Cluster-30738.137608, Cluster-30738.103058, Cluster-30738.134573, Cluster-167128.2, and Cluster-30738.125201 began to increase significantly in the process of hatching from eggs to larva. After being engorged, it showed a downward trend, and then showed an upward trend in the process of developing to the next stage of unfed. After that, this wavy mode of expression continued until fed adult. Meanwhile, the other class of cuticle had seven members, namely, Cluster-699.0, Cluster-30738.145384, Cluster-30738.187998, Cluster-30738.172572, Cluster-30738.167454, Cluster-30738.145385, and Cluster-30738.165925, which showed a downward trend in the process of egg development to unfed larva. Then, in the subsequent development process, it showed a significant upward trend from unfed stage to the corresponding fed stage and a significant downward trend in the process from the fed stage to the next unfed stage (Table 7) .
Table 7
Protein | ID | Mean_Ratio HLUL-VS-HLEE | Mean_Ratio HLFL-VS-HLUL | Mean_Ratio HLUN-VS-HLFL | Mean_Ratio HLFN-VS-HLUN | Mean_Ratio HLUA-VS-HLFN | Mean_Ratio HLFA-VS-HLUA |
Cuticle protein, putative [Ixodes scapularis] | Cluster-30738.125201 | 4.29 | 0.55 | 2.44 | 0.45 | 3.2 | 0.37 |
Cuticle protein, putative [Ixodes scapularis] | Cluster-30738.137608 | 1.22 | 1.06 | 1.29 | 0.88 | 1.51 | 0.96 |
Cuticle protein, putative [Ixodes scapularis] | Cluster-30738.103058 | 2.16 | 0.64 | 2.65 | 0.34 | 2.05 | 0.57 |
Cuticle protein, putative [Ixodes scapularis] | Cluster-30738.134573 | 3.23 | 0.88 | 2.01 | 0.49 | 1.97 | 0.45 |
Cuticle protein, putative [Ixodes scapularis] | Cluster-699.0 | 0.81 | 1.32 | 0.85 | 2.33 | 0.43 | 6.79 |
Cuticle protein, putative [Ixodes scapularis] | Cluster-30738.145384 | 0.59 | 9.2 | 0.21 | 4.2 | 0.16 | 2.11 |
Cuticle protein, putative [Ixodes scapularis] | Cluster-30738.187998 | 0.63 | 1.55 | 1.28 | 1.44 | 0.68 | 9.31 |
Cuticle protein, putative [Ixodes scapularis] | Cluster-30738.172572 | 0.76 | 1.58 | 0.58 | 1.86 | 0.67 | 10 |
Cuticle protein, putative [Ixodes scapularis] | Cluster-167128.2 | 1.16 | 0.82 | 1.37 | 0.71 | 2.16 | 0.49 |
Cuticle protein, putative [Ixodes scapularis] | Cluster-30738.167454 | 0.83 | 4.28 | 0.41 | 2.88 | 0.29 | 2.3 |
cuticle protein, putative [Ixodes scapularis] | Cluster-30738.145385 | 0.5 | 7.85 | 0.22 | 5.51 | 0.17 | 1.61 |
cuticle protein, putative [Ixodes scapularis] | Cluster-30738.165925 | 1.34 | 2.82 | 0.43 | 1.88 | 0.47 | 1.11 |
cuticle protein, putative [Ixodes scapularis] | Cluster-30738.125201 | 4.29 | 0.55 | 2.44 | 0.45 | 1.14 | 0.37 |
*HLEE, egg; HLUL, unfed larva; HLFL, fed larva; HLUN, unfed nymph; HLFN, fed nymph; HLUA, unfed adult; HLFA, fed adult. |
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 classification, 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 first class, Cluster-30738.172187, was relatively steady at different developmental stages, and there was no obvious stage specificity. The second class, Cluster-30738.179201, Cluster-30738.171068, Cluster-30738.174970, Cluster-30738.209558, Cluster-30738.172113, and Cluster-30738.171857, showed a significant upward trend from egg development to unfed larva, and then decreased significantly from unfed stage to the corresponding fed stage, while its expression abundance increased significantly from the fed stage to the next unfed stage. The third class, Cluster-30738.179418, Cluster-30738.180943, Cluster-30738.180943, Cluster-30738.77125, and Cluster-30738.172989, had a common pattern, and its expression abundance increased significantly from the unfed stage to the corresponding fed stage and reached a peak at the fed adult (Table 8).
Table 8
Protein | ID | Mean_Ratio HLUL-VS-HLEE | Mean_Ratio HLFL-VS-HLUL | Mean_Ratio HLUN-VS-HLFL | Mean_Ratio HLFN-VS-HLUN | Mean_Ratio HLUA-VS-HLFN | Mean_Ratio HLFA-VS-HLUA |
Peritrophic membrane chitin-binding protein, putative [Ixodes scapularis] | Cluster-30738.190566 | 0.58 | 2.69 | 0.52 | 2.07 | 0.3 | 3.4 |
Integral membrane protein, putative [Ixodes scapularis] | Cluster-30738.172187 | 0.85 | 0.97 | 1.03 | 1.04 | 1.15 | 0.99 |
PREDICTED: mitochondrial import inner membrane translocase subunit TIM14-like isoform X2 [Octopus bimaculoides] | Cluster-30738.179418 | 0.79 | 1.4 | 0.74 | 1.38 | 0.72 | 1.57 |
TPA_inf: membrane protein [Amblyomma variegatum] | Cluster-30738.179201 | 1.54 | 0.93 | 1.29 | 0.92 | 1.29 | 2.22 |
Basement membrane-specific heparan sulfate proteoglycan core protein, partial [Stegodyphus mimosarum] | Cluster-30738.171068 | 1.31 | 1.43 | 1.15 | 0.84 | 1.49 | 0.93 |
antigen B membrane protein, putative [Ixodes scapularis] | Cluster-30738.180943 | 0.74 | 4.41 | 0.42 | 3.39 | 0.26 | 2.09 |
membrane protein, putative [Ixodes scapularis] | Cluster-30738.174970 | 1.64 | 0.78 | 1.69 | 0.68 | 1.38 | 0.71 |
antigen B membrane protein, putative [Ixodes scapularis] | Cluster-30738.180943 | 0.74 | 4.41 | 0.42 | 3.39 | 0.26 | 2.09 |
PREDICTED: plasma membrane calcium-transporting ATPase 2-like, partial [Cyprinodon variegatus] | Cluster-30738.209558 | 1.64 | 0.52 | 1.75 | 0.85 | 1.39 | 0.71 |
membrane-bound O-acyltransferase domain-containing protein 2 [Parasteatoda tepidariorum] | Cluster-30738.172113 | 1.3 | 1.03 | 1.05 | 0.73 | 1.21 | 1.49 |
Vesicle-associated membrane protein 1, partial [Stegodyphus mimosarum] | Cluster-30738.171857 | 3.17 | 0.53 | 2.11 | 0.45 | 3.66 | 0.1 |
peritrophic membrane chitin binding protein, putative [Ixodes scapularis] | Cluster-30738.77125 | 1.19 | 1.71 | 0.65 | 2.85 | 0.23 | 2.41 |
adipocyte plasma membrane-associated protein, putative [Ixodes scapularis] | Cluster-30738.172989 | 1.15 | 1.78 | 0.99 | 1.08 | 0.68 | 1.16 |
*HLEE, egg; HLUL, unfed larva; HLFL, fed larva; HLUN, unfed nymph; HLFN, fed nymph; HLUA, unfed adult; HLFA, fed adult. |
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 five salivary gland-associated proteins were identified in this experiment, and they were classified into two classes according to their expression patterns in seven different developmental stages. The expression abundance of the first 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 significantly during the development from unfed nymphs to fed adult (Table 9).
Table 9
Protein | ID | Mean_Ratio HLUL-VS-HLEE | Mean_Ratio HLFL-VS-HLUL | Mean_Ratio HLUN-VS-HLFL | Mean_Ratio HLFN-VS-HLUN | Mean_Ratio HLUA-VS-HLFN | Mean_Ratio HLFA-VS-HLUA |
Secreted salivary gland peptide, putative [Ixodes scapularis] | Cluster-30738.172529 | 0.91 | 1.53 | 0.8 | 1.4 | 0.72 | 6.98 |
Salivary protein Sal4 [Rhipicephalus | Cluster-30738.164072annulatus | 5.75 | 0.9 | 1.04 | 0.91 | 1.94 | 0.18 |
salivary sulfotransferase, putative [Ixodes scapularis] | Cluster-30738.173721 | 0.72 | 1.78 | 0.49 | 2.8 | 0.27 | 3.85 |
TPA_inf: salivary gland protein 223 [Amblyomma variegatum] | Cluster-30738.175111 | 0.84 | 1.43 | 0.86 | 2.98 | 0.35 | 3.87 |
TPA_inf: secreted salivary protein 1024 [Amblyomma variegatum] | Cluster-30738.171953 | 1.02 | 2.05 | 1.12 | 0.94 | 2.15 | 0.56 |
*HLEE, egg; HLUL, unfed larva; HLFL, fed larva; HLUN, unfed nymph; HLFN, fed nymph; HLUA, unfed adult; HLFA, fed adult. |
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 identified, and many proteins also showed a strong regularity and specific up-regulated expression at different developmental stages. These secreted proteins were mainly divided into three classes by collating the data. The first class, including a total of 12 proteins (Cluster-30738.87074, Cluster-30738.200760, Cluster-30738.71323, Cluster-30738.173742, Cluster-30738.63453, Cluster-175252.0, Cluster-30738.196486, Cluster-30738.177356, Cluster-30738.174134, Cluster-30738.182142, Cluster-30738.149846, and Cluster-30738.173637), showed a significant growth trend from the eggs stage hatched into unfed larva. After that, its expression abundance also showed a significant growth trend in the process from the fed stage to the next unfed stage. The second class, which consisted of 11 proteins (Cluster-30738.40780, Cluster-30738.172492, Cluster-30738.173758, Cluster-30738.236193, Cluster-30738.4675, Cluster-30738.168968, Cluster-30738.173651, Cluster-30738.173029, Cluster-30738.170909, Cluster-30738.173549, and Cluster-30738.172529), seemed to have some relationship with the process of satiety, and the expression of these proteins was significantly up-regulated in the starvation stage and the corresponding satiety stage and reached the peak at the stage of engorged adult. In the third class, including a total of 15 proteins, the regularity of these proteins seemed to be closer to the specificity of each developmental stage of H. longicornis, mainly including Cluster-30738.81824, Cluster-30738.201894, Cluster-30738.171064, Cluster-30738.236147, Cluster-30738.92518, Cluster-30738.58441, Cluster-30738.179348, Cluster-30738.171953, Cluster-30738.170773, Cluster-30738.176166, Cluster-30738.4778, Cluster-30738.170966, Cluster-30738.169678, and Cluster-30738.160134 (Table 10).
Table 10
Protein | ID | Mean_Ratio HLUL-VS-HLEE | Mean_Ratio HLFL-VS-HLUL | Mean_Ratio HLUN-VS-HLFL | Mean_Ratio HLFN-VS-HLUN | Mean_Ratio HLUA-VS-HLFN | Mean_Ratio HLFA-VS-HLUA |
secreted protein, putative [Ixodes scapularis] | Cluster-30738.87074 | 1.25 | 1.19 | 1.73 | 0.49 | 2.47 | 0.51 |
secreted protein, putative [Ixodes scapularis] | Cluster-30738.81824 | 0.79 | 0.29 | 9.8 | 0.23 | 7.77 | 0.1 |
secreted protein, putative [Ixodes scapularis] | Cluster-30738.200760 | 3.11 | 0.93 | 2.39 | 0.69 | 1.95 | 0.44 |
secreted protein, putative [Ixodes scapularis] | Cluster-30738.201894 | 2.31 | 0.91 | 1.12 | 1.19 | 0.7 | 1.47 |
secreted protein, putative [Ixodes scapularis] | Cluster-30738.40780 | 0.68 | 1.45 | 0.54 | 1.65 | 0.57 | 2.11 |
secreted salivary gland peptide, putative [Ixodes scapularis] | Cluster-30738.171064 | 1.02 | 1.44 | 0.98 | 1.18 | 1.08 | 0.86 |
secreted salivary gland peptide, putative [Ixodes scapularis] | Cluster-30738.236147 | 0.65 | 0.57 | 5.42 | 0.53 | 4.73 | 0.55 |
secreted salivary gland peptide, putative [Ixodes scapularis] | Cluster-30738.71323 | 5.5 | 0.6 | 2.14 | 0.52 | 2.08 | 0.45 |
secreted cysteine rich protein, putative [Ixodes scapularis] | Cluster-30738.173742 | 3.36 | 0.41 | 2.49 | 0.19 | 10 | 0.1 |
secreted protein, putative [Ixodes scapularis] | Cluster-30738.172492 | 0.67 | 2 | 0.41 | 1.15 | 0.96 | 1.44 |
secreted salivary gland peptide, putative [Ixodes scapularis] | Cluster-30738.173758 | 0.88 | 1.32 | 0.74 | 3.13 | 0.38 | 1.92 |
secreted protein, putative [Ixodes scapularis] | Cluster-30738.63453 | 2.44 | 0.55 | 1.98 | 0.88 | 1.09 | 0.64 |
secreted protein, putative [Ixodes scapularis] | Cluster-175252.0 | 2.41 | 0.73 | 1.02 | 0.85 | 1.03 | 0.94 |
secreted salivary gland peptide, putative [Ixodes scapularis] | Cluster-30738.92518 | 0.64 | 2.01 | 1.66 | 0.68 | 1.75 | 0.88 |
secreted salivary gland peptide, putative [Ixodes scapularis] | Cluster-30738.236193 | 0.35 | 2.85 | 0.26 | 2.99 | 0.78 | 2.05 |
secreted salivary gland peptide, putative [Ixodes scapularis] | Cluster-30738.4675 | 0.86 | 1.81 | 0.83 | 2.93 | 0.33 | 4.13 |
secreted protein, putative [Ixodes scapularis] | Cluster-30738.168968 | 1.12 | 1.2 | 0.51 | 2.52 | 0.5 | 3.2 |
secreted protein, putative [Ixodes scapularis] | Cluster-30738.173651 | 0.8 | 2.19 | 0.77 | 1.6 | 0.7 | 1.25 |
TPA_exp: hypothetical secreted protein 1652 [Amblyomma variegatum] | Cluster-30738.196486 | 1.29 | 1.2 | 1.96 | 0.56 | 1.89 | 0.57 |
putative secreted protein [Ixodes scapularis] | Cluster-30738.58441 | 1.23 | 1.66 | 0.77 | 1.03 | 1.11 | 0.81 |
secreted salivary gland peptide, putative [Ixodes scapularis] | Cluster-30738.173029 | 0.79 | 1.06 | 0.8 | 1.6 | 0.93 | 3.04 |
putative secreted salivary gland peptide [Ixodes scapularis] | Cluster-30738.177356 | 1.72 | 0.94 | 2.88 | 0.46 | 2.59 | 0.22 |
secreted salivary gland peptide, putative [Ixodes scapularis] | Cluster-30738.170909 | 1.01 | 1.79 | 0.59 | 2.2 | 0.37 | 1.29 |
TPA_inf: putative secreted glycine-rich protein [Amblyomma variegatum] | Cluster-30738.174134 | 1.47 | 1.33 | 1.27 | 0.87 | 1.65 | 0.68 |
secreted protein, putative [Ixodes scapularis] | Cluster-30738.179348 | 0.43 | 0.57 | 0.94 | 2 | 0.92 | 1.7 |
TPA_inf: secreted salivary protein 1024 [Amblyomma variegatum] | Cluster-30738.171953 | 1.02 | 2.05 | 1.12 | 0.94 | 2.15 | 0.56 |
secreted protein, putative [Ixodes scapularis] | Cluster-30738.170773 | 0.7 | 4.1 | 0.31 | 1.46 | 0.57 | 2.07 |
glycine proline-rich secreted protein, putative [Ixodes scapularis] | Cluster-30738.176166 | 1.4 | 0.84 | 0.86 | 0.83 | 1.71 | 1.4 |
secreted salivary gland peptide, putative [Ixodes scapularis] | Cluster-30738.173549 | 0.69 | 4.01 | 0.39 | 4.32 | 0.16 | 6.96 |
secreted protein, putative [Ixodes scapularis] | Cluster-30738.4778 | 1.02 | 1.88 | 1.15 | 0.76 | 1.5 | 0.62 |
Secreted salivary gland peptide, putative [Ixodes scapularis] | Cluster-30738.170966 | 0.6 | 1.19 | 0.66 | 0.86 | 1.12 | 0.86 |
TPA_inf: hypothetical secreted protein 790 [Amblyomma variegatum] | Cluster-30738.169678 | 0.96 | 1.3 | 1.08 | 0.84 | 1.94 | 0.65 |
Secreted protein, putative [Ixodes scapularis] | Cluster-30738.182142 | 1.26 | 1.32 | 1.62 | 0.72 | 1.35 | 0.59 |
Secreted protein, putative [Ixodes scapularis] | Cluster-30738.149846 | 1.54 | 0.87 | 1.37 | 0.89 | 1.13 | 0.86 |
secreted salivary gland peptide, putative [Ixodes scapularis] | Cluster-30738.172529 | 0.91 | 1.53 | 0.8 | 1.4 | 0.72 | 6.98 |
TPA_inf: hypothetical secreted protein 790 [Amblyomma variegatum] | Cluster-30738.160134 | 0.59 | 0.52 | 2.42 | 0.74 | 2.54 | 0.31 |
glycine proline-rich secreted protein, putative [Ixodes scapularis] | Cluster-30738.173637 | 1.64 | 1.28 | 1.73 | 0.63 | 1.73 | 0.27 |
*HLEE, egg; HLUL, unfed larva; HLFL, fed larva; HLUN, unfed nymph; HLFN, fed nymph; HLUA, unfed adult; HLFA, fed adult. |
GO analysis of the DEPs
Functional classification of the DEPs was carried out through GO analysis. We have identified 41, 45, 41, 44, 44, and 45 GO terms respectively in the HLUL vs. HLEE, HLFL vs. HLUL, HLUN vs. HLFL, HLFN vs. HLUN, HLUA vs. HLFN, and HLFA vs. HLUA groups (HLEE, egg; HLUL, unfed larva; HLFL, fed larva; HLUN, unfed nymph; HLFN, fed nymph; HLUA, unfed adult; HLFA, fed adult). Among these GO terms, there were 19 biological process GO items, 14 cell component GO items, and eight molecular function GO items in UL vs. EE. The GO terms in the FL vs. UL included 23 biological process terms, 14 cell component terms, and eight molecular function terms. The GO terms in the UN vs. FL included 19 biological process terms, 14 cell component terms, and eight molecular function terms. In FN vs. UN, there were 20 biological process terms, 14 cell component terms, and eight molecular function terms. For the GO terms in the UA vs. FN, there were 22 biological process terms, 14 cell component terms, and eight molecular function terms. The GO terms in FA vs. UA included 23 biological process terms, 14 cell component terms, and eight molecular function terms. In order to further explore the functions and properties of the up-regulated and down-regulated proteins in the different developmental stages of H. longicornis, we performed clustering and abundance analyses of these GO terms. The figure shows up to 20 rich GO terms in each group and up to three main GO cluster graphs (Figures 4 and 5).
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. longicornis, the HLUL vs. HLEE, HLFL vs. HLUL, HLUN vs. HLFL, HLFN vs. HLUN, HLUA vs. HLFN, and HLFA vs. HLUA groups (HLEE, egg; HLUL, unfed larva; HLFL, fed larva; HLUN, unfed nymph; HLFN, fed nymph; HLUA, unfed adult; HLFA, fed adult) were analyzed and identified 2,112; 2,124; 4323; 3,371; 2,846; and 1,998 channels, respectively (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.