The P-fam protein family were annotated with antithrombin collected by querying the interproscan website [13, 14]. All genes annotated to the same P-fam family were then screened as anticoagulant-related genes in H. manillensis. The results showed that there were 155 anticoagulant-related gene sequences in the H. manillensis genome. These genes were categorized using anticoagulant and anticoagulant-related gene types (analgesic and anti-inflammatory, inhibition of platelet, extracellular matrix degradation, and direct anticoagulation). The results are shown in Table 4. The number of anticoagulant and anticoagulant-related loci in H. manillensis suggests the possible anticoagulant effects and medicinal value.
Table 4
Classification of anticoagulant and anticoagulant genes in H. manillensis
Functional classifications
|
Gene description
|
Obtained gene number
|
Analgesic and anti-inflammatory
|
Antistasin family
|
34
|
Interleukin-17
|
2
|
Kunitz/Bovine pancreatic trypsin inhibitor domain
|
6
|
Hemerythrin HHE cation binding domain
Macin
|
3
2
|
Spider toxin
|
1
|
Potato inhibitor I family
|
17
|
Extracellular matrix degradation
|
Inter-alpha-trypsin inhibitor heavy chain C-terminus
Matrixin
|
1
21
|
Inhibition of platelets
|
Apyrase
|
8
|
Destabilase
Disintegrin
|
8
51
|
Anticoagulant
|
Hirudin
|
1
|
Note: Functional classification: Functional annotation classification; Obtained gene number: Number of genes belonging to this classification
Anticoagulant-related genes of H. manillensis
Antistasin family-related genes
The antistasin family of proteins is a class of cysteine-rich serine protease inhibitors that can markedly inhibit the coagulation process, albeit through different mechanisms of action [15-17]. Antistasin can also respond to host inflammatory responses, exerting an anti-inflammatory effect. Among the genes related to the antistasin family identified in this study, 10 related genes were upregulated and 5 were downregulated upon blood sucking in H. manillensis. This indicates that the antistasin protein family plays an important role in the anticoagulant process after the blood sucking of H. manillensis.
ATP diphosphate hydrolase (apyrase)-related genes
ATP diphosphate hydrolase (apyrase) is an antiplatelet factor. The mechanism by which apyrase in the saliva of bloodsucking arthropods works is as follows: ADP present in the host after tissue damage promotes platelet activation, but apyrase can hydrolyze ATP and ADP to AMP and inorganic phosphorus, which hinders platelet aggregation [18-22]. This process makes it difficult for a bitten host to form a thrombus, which facilitates the feeding of bloodsucking animals.
In this study, three apyrase-related genes were shown to be downregulated after blood sucking by H. manillensis and one was downregulated. This indicates that apyrase participates in the anticoagulation process after H. manillensis bloodsucking.
Destabilase-related genes
Destabilase acts as a thrombolytic agent in leech; it cleaves peptide bonds and promotes fibrinolysis, thereby inhibiting coagulation activity [23-26]. As a multifunctional enzyme, destabilase can also exert antibacterial function. In addition, the salivary glands of the leech can also secrete other kinds of enzyme, which together with destabilase play a bacteriostatic role.
This study identified three genes related to destabilase that were upregulated after the blood sucking of H. manillensis and two that were downregulated. The results show that destabilase plays a role in the anticoagulant and antibacterial processes upon the sucking of blood by H. manillensis.
Disintegrin-related genes
Disintegrin contains a sperm-glycoside-aspartic acid sequence or a lysine-aspartate sequence and is rich in cysteine. It binds to the glycoprotein fibrinogen receptor on the platelet membrane, competitively antagonizing the binding of fibrin to platelets, and prevents platelet activation and changes in glycoprotein receptor conformation, blocking the binding of receptors to multiple ligands. In this way, it inhibits the final common pathway of platelet aggregation and blocks the formation of a thrombus.
This study identified 15 genes related to disintegrin that were upregulated after blood sucking by H. manillensis and 9 that were downregulated. This indicates that disintegrin plays an important role in the anticoagulation process after the blood sucking of this species.
Gene analysis of bloodsucking characteristics of H. manillensis
Finally, 155 genes related to anticoagulation were screened, corresponding to 13 active substances, including antistasin, apyrase, destabilase, and disintegrin. According to their functions and effects, these genes are classified into those involved in platelet inhibition and those directly encoding anticoagulants. Among them, the expression level of the antistasin family, which has both anticoagulant and anti-inflammatory effects, was generally increased. In terms of the expression trends of genes related to the inhibitors of platelet aggregation apyrase, destabilase, and disintegrin, these were upregulated. At the same time, the expression levels of related genes involved in the anti-inflammatory response of host wounds also showed an upward trend. This shows that, in the process of blood sucking, H. manillensis produces a variety of active anticoagulant substances, and directly or indirectly, these together inhibit the host’s blood coagulation process, accompanied by an anti-inflammatory response. Taken together, these findings indicate that the anticoagulant process is a complex biological pathway in which multiple substances cooperate and interact.
Upon consuming blood, bloodsucking animals can also take in infectious bacteria from the surface of the wound and immune proteins from the blood of the host. Therefore, they need to effectively protect themselves against bacterial invasion and immune protein attack. Destabilase has a strong inhibitory effect on bacteria. Hyaluronidase also acts as an active bacteriostatic protein, which dissolves the envelope of bacteria and forms antibodies, effectively inhibiting the activity of various bacteria and fungi. Some of the functional genes associated with the immune response, such as those annotated as hyaluronidase, were shown to be upregulated after the blood sucking of H. manillensis .
After bloodsucking animals ingest host blood, the hemoglobin in the blood tends to hydrolyze to produce a large amount of hemoglobin. Heme is a potent oxidant that produces hydroxyl radicals with excellent oxidizing ability. Studies have shown that heme can cause DNA damage in mitochondria and alter the expression of apoptotic proteins in these organelles. Hemoglobin also causes a certain degree of damage to the genome. In H. manillensis, hemoglobin is likely to cause great damage during the process of digesting blood. Therefore, this species needs to cope with the oxidative stress generated during blood digestion. Citelli et al. [15] asserted that a variety of proteins with oxidoreductase activity, such as superoxide dismutase, glutathione S-transferase, and thioredoxin, are involved in this antioxidant mechanism. In the process of blood sucking by animals such as ticks, the stress response and non-specific immunity play important roles. After the blood sucking of H. manillensis, the expression of related genes involved in the antioxidant process was found to be upregulated and the antioxidant capacity was significantly enhanced.
Upon blood sucking, H. manillensis needs to produce a large amount of active anticoagulant substances to ensure the fluidity of the host blood. At the same time, it also produces antibacterial and immune-related substances to protect against foreign substances, and proteins having oxidoreductase activity are produced by antioxidation to cope with oxidative stress generated by hemoglobin during blood digestion.
Functional analysis of KEGG pathway in anticoagulation-related DEGs
We found 155 anticoagulant-related genes based on the transcriptome data of H. manillensis and explored their related pathways. The Notch signaling pathway, nicotinate, and nicotinamide metabolism associated with anticoagulant genes are involved in cell growth. It is of great significance to help to detect the occurrence and development of tumors and to treat them in a targeted manner by excavating these pathways.
(1) Notch signaling pathway
In the KEGG database annotation, there are 48 Unigene annotations in the Notch signaling pathway, with three DEGs participating in the pathway. The Notch signaling pathway is important for cell-to-cell communication, which regulates cell differentiation, apoptosis, proliferation, and morphogenesis. It is also critical for the growth and development of organisms [27-32]. The Notch signaling pathway is mainly composed of Notch-related receptors, Notch-associated ligands, Notch downstream signal transduction molecules, and nuclear response factors. TACE (ADAM17) is a key enzyme in the activation of the Notch signaling pathway and has a significant limiting effect on its activation [33-35, 39]. Simultaneous activation of ADAM17 induces GPIbα digestion [36], and platelet membrane glycoprotein (GP) Ibα digestion reduces the expression of platelet surface functional receptors, resulting in weakened platelet adhesion. This affects the formation of blood clots and inhibits the aggregation function of platelets, being a recognized negative regulatory mechanism of platelets. After the feeding of H. manillensis , the expression of TACE-related gene (ADAM 17-like protease) is upregulated, which inhibits platelet aggregation, blocks thrombus formation, affects the activation of the Notch signaling pathway, and participates in the anticoagulant process.
Notch-mediated signaling plays a key role in the development of the cardiovascular system and cardiovascular disease, and is closely related to the regulation of immune system function, pancreatic cancer, and medulloblastoma [37-39]. Notch signaling may play different roles in different environments, times, or cell lines, and its over- or under-expression may have a negative impact on the organism. Therefore, an in-depth understanding of Notch signaling and its interaction with various pathways can provide a foundation for more research on the treatment of cardiovascular diseases and tumors, and for carrying out targeted and efficient clinical treatment.
(2) Nicotinate and nicotinamide metabolism
ATP diphosphate hydrolase (Apyrase) blocks platelet aggregation. The apyrase-related gene detected in this study is involved in the nicotinate and nicotinamide metabolism pathway, and for three enzymes there was upregulated gene expression after vaccination with H. manillensis, while for one enzyme there was downregulated gene expression.
In the KEGG database annotation, there are 27 Unigene annotations on the Notch signaling pathway, with four DEGs participating in the pathway. Nicotinamide phosphoribosyltransferase (Nampt) is the rate-limiting enzyme of this pathway [40-42]; it also has physiological functions such as promoting angiogenesis, anti-apoptosis, participating in the body’s inflammatory response, and promoting the proliferation, differentiation, and maturation of various cells. After the feeding of H. manillensis, apyrase-related genes are involved in the anticoagulant process, inhibiting platelet aggregation, and downregulating the expression of Nampt-related genes, affecting the niacin and nicotinamide metabolic pathways.
The concentration of Nampt in the blood also significantly increases in many tumor patients, so an inhibitor of the activity of this enzyme is an anti-tumor drug with great potential for clinical application [41-43]. However, in this context, there are still many problems to be resolved. The role of anticoagulation genes related to H. manillensis before and after blood sucking is discussed to provide a basis for the better application of tumor treatment.
SNP analysis
Single-nucleotide polymorphism(SNP)is mainly a DNA sequence polymorphism caused by DNA replication, a genetic marker formed by a single-nucleotide variation in the genome. These variations have become commonly used as molecular markers, and in disease diagnosis, paternity testing, plant breeding, and genetic mapping [44, 45]. By identifying potential SNP sites, it is possible to analyze whether these sites affect the expression level of a gene of interest or the particular protein that is produced.
SNPs can be divided into two types: transitions and transversions. Depending on the number of alleles at the SNP site, the SNP can be divided into a homozygous SNP site (only one allele) or a heterozygous SNP site (two or more alleles). There is a difference in the proportion of heterozygous SNPs in different species. SNP molecular markers were developed from H. manillensis transcriptome data. In the obtained 17,051 Unigenes of H. manillensis transcripts, an average of 154,399 SNP sites were found, of which 109,917 were transitions, accounting for 71.19%, and 44,482 were transversions, accounting for 28.81% (Table 5).
Table 5
Sample ID
|
SNP Number
|
Genic SNP
|
Intergenic SNP
|
Transition
|
Transversion
|
Heterozygosity
|
ST1
|
147115
|
124889
|
22226
|
71.41%
|
28.59%
|
51.76%
|
ST2
|
169399
|
140997
|
28402
|
70.70%
|
29.30%
|
49.96%
|
ST3
|
132107
|
111179
|
20928
|
71.54%
|
28.46%
|
51.54%
|
AF1
|
194328
|
159091
|
35237
|
70.21%
|
29.79%
|
52.70%
|
AF2
|
173142
|
145623
|
27519
|
70.70%
|
29.30%
|
51.13%
|
AF3
|
110302
|
94053
|
16249
|
72.57%
|
27.43%
|
47.29%
|
Note:Sample ID:Sample analysis number; SNP Number: Total number of SNP sites; Genic SNP: Total number of SNP sites in the gene region; Intergenic SNP: Total number of SNP sites in the intergenic region; Transition: The percentage of the number of conversion-type SNP sites within the total number of SNP sites; Transversion: The percentage of the number of SNP sites of the transversion type within the total number of SNP sites; Heterozygosity: The percentage of heterozygous SNP sites in the total number of SNP sites.