Ticks are of vast medical and veterinary public health importance due to direct damage in livestock by its hematophagous feeding habits and its potential as a vector capable to transmit infectious agents such as Tick-borne diseases. Currently, the knowledge of vertebrates’ immune system contributes to the advance in vaccine and drug development, resulting in new drugs that help to control human and livestock pathogens. Unfortunately, very small advances have been achieved in tick’s immune system that could help to develop new strategies designated to control tick-borne diseases and other arthropod vectors. On this subject, the study of the mechanisms involved is transcendental as is also the study on molecules, cells, and regulation of immune response involved in signaling pathways in ticks. The progress on the understanding of ticks’ physiology represents a necessary advance in molecular approaches related with a tick’s immune response, involved in host-vector-pathogen interaction, and, in turn, evolutionary relationships. Current knowledge on tick’s immune response to different kinds of pathogens is described in this chapter and the use of modern molecular tools to fill the gaps on different aspects in tick immunobiology that still is unclear or under study.
- immune system
Ixodidae, comprising those arthropods commonly called ticks, include nearly 870 acaridae species, and these are obligate hematophagous parasites of terrestrial vertebrates at some part of their life cycle. Moreover, ticks are considered as important veterinary health threat, due to their capacity to cause direct damage to livestock by feeding on blood and transmitting tick-borne pathogens, causing serious animal and human infectious diseases. The pathogenic diversity of organisms transmitted by ticks exceeds to be found in all other hematophagous arthropods. Tick-borne pathogens include protozoans, the bacteria rickettsia, viruses, and nematodes  that in turn evade the tick’s immune defense mechanisms, encountered on their route through the tick’s body (midgut, hemolymph, salivary glands or ovaries). These immune interactions are very important in tick biology and pathogen relationships. Likewise, some pathogens are also often trans-stadia and trans-ovarian transmitted increasing the vector-pathogen complexity, related with a disease transmission and severity, considering that each tick species is capable to transmit different pathogens . Unfortunately, many metabolic and molecular mechanisms related with a tick’s immune response to different pathogens remain unclear. For this reason, in recent years, the study of tick-host-pathogen interface has increased. Currently, we know that tick’s innate immunity is carried on by the cellular and innate responses, where the different molecules, enzymes, cells, and proteins are involved in general immune mechanism. On the other hand, we have different molecular and immunological tools, as tick’s salivary glands, midgut transcriptome, and proteomics analysis, and the first tick genome project, that contribute to elucidate tick’s biological interactions. The immunobiology characterization of the tick-pathogen-host interface dynamic interaction should be exploited as a tool used for development of novel vector and transmission blocking vaccines, targets, and new drug design [3, 4].
2. Immune system in invertebrates
All invertebrates have an immune system, composed of both humoral and cellular response that results as effective defense to different pathogen attack. The cellular immune response is composed of several mechanisms as phagocytosis, nodulation formation, agglutination, and cellular encapsulation, while humoral response involves expression and secretion of different molecules able to kill bacteria, parasites, and other pathogens [2, 4]. The performance of this multifactorial system requires synthesis and regulation of RNA and proteins involved in arthropod protection. Until recently, the investigations of molecular, genetics, and cellular aspects of the arthropods’ immune response were scant. One reason behind this paucity is the extremely difficult to control laboratory conditions that allow to maintain the host-parasite interaction in several generations, or different stages of arthropod life cycle . On the other hand, among the invertebrates, the insects have received most attention, compared to arachnids. In this regard, in spite of extensive research, the immune system of ticks is still poorly understood.
2.1. Ticks’ immunobiological response
The immune response of ticks as well as arthropods includes both cellular and humoral mechanisms, where the hemolymph and other tissues, such as salivary glands, midgut, hemolymph, and fat body, provide the principal source of molecules and cells involved in the immunological attack of pathogens. In the case of tick’s hemolymph, many pathways involved in the immune response still remain unclear . Currently, few reports explain the type of response that ticks have against different infectious agents that, in turn, could be used as target to pest biocontrol.
3. Cellular immune response
All tissues in ticks, and other invertebrates, are bathed in a fluid known as the hemolymph, which is the first source of nutrients, osmoregulation, and molecules and hormones transport, and provide protection to pathogen agents to which ticks are exposed . Likewise, hemolymph coagulates at the site of some injury, preventing microbes spreading into the body tissues. The hemolymph consists of protein-rich plasma and different types of cells called hemocytes that play a transcendental role in immune response .
In ticks, the immune cell–mediated response is carried out by hemocytes, cells with free circulation and the major component of the hemolymph. Hemocytes play an important role in the tick’s defense against injury as well as microbial infection and increase greatly its population, in response to bacteria, viruses, protozoa, and other pathogen infection; however, the multiplication rates for the hemocyte types in response to a specific pathogen have not been fully clarified [5, 6, 7, 8]. The mature hemocytes mediate different events that include phagocytosis, nodulation, and encapsulation. The tick hemolymph can be divided into four cell types based on their function and morphology; however, at the moment, the hemocytes classification is controversial, because it has been observed that population may varied in hard and soft ticks and among species. The prohemocytes are round to oval small cells with a prominent nucleus, numerous mitochondria, and little granular cytoplasm. The cell size is 6–7 μm and represents the stem cells in the hemolymph, from which all cell types can be differentiated and occasionally can be found to be associated with many tissues. The prohemocytes’ population proportion varies depending on the species and healthy, wounded, or infected ticks [9, 10]. The granulocytes are large cells with numerous cytoplasmatic granules; some cases have a cytoplasmatic extension called filopodia. In general, granulocytes have a long size about 15–20 μm and are further subdivided into type I and type II, depending on the granule morphology. Type I granulocytes are pleomorphic cells that 6 μm in length, which contain variable electrodense granules and presence of filopodia and lysosomes. The type II granulocytes contain several granules both electrodense and condensing immature granules, located peripherally and at the central cell . Along with granulocytes, the plasmatocytes are the most predominant hemocyte type in hemolymph. These cells have slightly elongated shape, often fusiform and numerous filopodia, with a large variability in size ranging from 8 to 12 and the long axis up to 20 μm. In some species, plasmatocytes have rounded or ovoid shape, with a size about 10–12 μm and containing few vacuoles and granules. The spherulocytes are cells with a size of 11–14 μm and are oval shaped with electron-lucent and fibril-filled granules that fill almost the entire cytoplasm cell. Currently, some studies report the presence of the oenocytoids in limited number of tick species . These cells are 11–18 μm in size and are ovoid shaped with cytoplasmatic granules . However, the oenocytoids’ presence in ticks remains controversial . The understanding of functions and pathways involved in the activation of hemocytes could provide elements that help to understand the cells’ role in immune response. In this regard, many groups have studies based on electrophoretic patterns in one and two dimension, obtaining proteomic maps that show proteins related with the hemocytes’ pathogen response .
Phagocytosis is a complex mechanism that involves the recognition, engulfment, and destruction of pathogens. In this process, the immune cells recognize pathogen-associated molecular patterns (PAMPs) produced by several bacteria and fungi. In all arthropods, phagocytosis is carried out by the hemocytes and represents the first primary defense response to pathogen infection [11, 14]. In ticks, the phagocytosis process has been regulated by granulocytes type I and plasmocytes and sometimes by granulocytes type II, suggesting that differences in hemocytes’ population have different roles and contributions to the tick’s immune response . In initial steps, the phagocytic cell response is binding receptor-mediated to pathogen cell surface; subsequently, signal transduction pathways are activated and followed by filopodia projections that surround and engulf the bound particle . The particle is internalized by endocytosis into a vesicle, subsequently, with lysosomal compartments that in turn form the phagolysosome. Inside, intracellular enzymes are activated such as acid phosphatases, type c lysozyme, cystatins, and proteases completing the cellular lysis. Little is known about the molecular regulation in tick immune response, some reports suggested that as in insects the most important signal transduction pathways are mitogen-activated protein kinase (MAPK) and FAK/Src pathways that in turn are involved in proPO activation . Moreover, several external factors are capable to enhance this process. Currently, recent evidence indicates that
Tick hemocytes are capable of expressing lectins on membrane surface involved in pathogen recognition. These molecules can join with lipopolysaccharides (LPS) also present on the pathogen surface. Currently, several lectins involved in the immune response and other mechanisms have been identified in tick hemocytes and different cells [18, 19, 20, 21]. In soft ticks,
Encapsulation is the immunological process whereby the arthropods are capable of attacking pathogens that are very large to eliminate by nodulation or phagocytosis. Other immunological processes, such as the proteolytic degradation of microbial products (LPS and peptidoglycan), can result in the prophenoloxidase activation. This activation generates phenoloxidase expression that in turn, along with tyrosine metabolism, is directly related to melanin synthesis. In all insects, pathogen encapsulation involves melanization, where hemocytes, mainly type I granulocytes and plasmatocytes, form a capsule of thick layer around the pathogen that leads to asphyxiation and toxic-free radical production, such as quinones and semiquinones [3, 26, 27], with melanin deposition as the final step . In ticks, phenoloxidase is present in
4. Humoral immune response
The humoral factors of the insect and crustacean immune system have been extensively studied. In contrast, in ticks, we know very little of this field. Mostly, the soluble factors are produced by hemocytes and released in the hemolymph, where they are transported to other tissues such as midgut and salivary glands. The humoral factors play an important role in the defense and protection of ticks from microbial invasion. Within these factors, a variety of antimicrobial proteins, such as lectins, proteases, and lysozymes, coagulation factors, proteases inhibitors, antimicrobial peptides, and products related to oxidative stress, are included [3, 32]. These soluble factors are involved in various aspects of the immune protection, such as blood ticks feeding in midgut protection; during migration hemolymph defense; and tissue protection, for example, during pathogen transmission in salivary glands, in all cases during pathogen infestation . The plasma hemolymph represents nearly 90% of total composition, and the proteic soluble component represents approximately 11.5–14.3% of plasma . The knowledge of ticks’ hemolymph components is very limited; for this reason, the advance in the understanding is based on other arthropods . For example, electrophoresis assays of two-dimensional gel map obtained of
4.1. Antimicrobial peptides (AMPs)
Antimicrobial peptides (AMPs) represent the most effective humoral immune response, for their ability to kill several pathogens, for their fast response, and for their effectiveness at micronanomolar concentrations. AMPs are small peptides (3–20 kDa), and their action mechanism is based on their capacity to cell membrane or cell wall binding, causing structural disruption that results in loss of pathogen membrane potential. AMPs are secreted mainly by the fat body and hemocytes; however, midgut is capable to produce some peptides . Many authors reported and identified several AMPs in ticks, including microplusin , hebraein , ixodidin , antimicrobial peptide (ISAMP) , and some peptides from
Defensins are small cationic peptides (3–6 kDa) with six to eight cystein residues that are folded by three or four disulfide bridges. These bonds help to stabilize and maintain the tertiary structure, called “defensin folds” . Defensin AMPs were found in many arthropods including hard and soft ticks [41, 42]. Defensins may be classified into three major groups: (1) peptides with α-helical conformation, (2) cyclic and open cyclic peptides with cysteine residue pairs, and (3) peptides with overrepresentation of some amino acids . In all cases, the mature peptides present highly conserved regions in contrast with leader regions that show much more variability. Moreover, its sequence contains hydrophobic regions separated from charged regions that enable them to insert into pathogen membranes causing pores that in turn kill the cell . In ticks, the defensin expression is carried out in several tissues such as fat body, hemocytes, salivary glands, and midgut.
Lysozymes are ubiquitously expressed enzymes with a molecular weight approximately 14 kDa, are involved in digestive processes, and have an antimicrobial activity for their ability to lyse bacteria by hydrolyzing the β-1,4 glycosidic bonds between the N-acetyl-muramic acid and N-acetyl-D-glucosamine residues that form the peptidoglycan walls. In a hard tick
4.1.3. Other antimicrobial peptides
Currently, in addition to defensins, there exist a large number of antimicrobial peptides identified. In ticks, other types of AMPs have been detected. In
4.2. Hemagglutination (lectins)
Lectins are proteins whose structure has domains with specific binding sites for carbohydrate . The bacteria membrane or cell wall including the fungi and protozoan pathogens has different carbohydrate moieties that can be recognized by lectins. These proteins exhibit different molecular sizes from 30 to 85 kDa and have been identified in the membrane surface of hemocytes, cell gut, and salivary glands, or synthesized by hemocytes and released in the hemolymph plasma of soft and hard tick species. In invertebrates, lectins are important mediators of immune response. Initially, these molecules are defined by their participation in a hemagglutination process; however, these proteins also bind to pathogens that, in turn, enable hemocytes to recognize and engulf (opsonization). This process includes carbohydrate recognition by ficolins and mannose-binding gal-lectins, among others [55, 56]. Insects, ascidians, crustaceans, and ticks contain molecule type TLP-1 and TLP-2 lectins (
4.3. Proteases and protease inhibitors
The feeding mechanisms of ticks involve the presence of midgut, where the blood mead digestion is carried out. In this process, a large variety of cysteine, aspartic, and serine proteases are involved, and many of these molecules also have an important role in mechanisms of immune response. In the lumen, the serine proteases are the most important, which function as hemolytic agents and as cysteine and aspartyl proteases in hemoglobin digestion [66, 67, 68]. Various of these proteins are identified; however, in ticks, the regulation, expression, and presence of these molecules still remain unclear. Currently, the protease immune mechanism in insects suggests that metalloproteases may be important in cellular immune defense . In the
4.3.2. Protease inhibitors
Protease inhibitors are important in tick’s pathogen infection as innate immune suppressor of virulence, toxic, and replication factors expressed by microorganisms. Proteases are important virulence factors used in various stages of the infection process, both by prokaryote and eukaryote pathogens. The inactivation of these factors may prevent the pathogen survival in the tick .Two major protease inhibitors have been reported in ticks: one called serpins that act as serine proteinase inhibitors and the other α-macroglobulins, large glycoproteins with mostly thiol-ester–containing proteinase inhibitors. Serpins may be found in plasma hemolymph and small cytoplasm granules ; however, in
5. Nitric oxide and oxidative stress
5.1. Nitric oxide synthase
The nitric oxide (NO) is an unstable radical, capable to act with a key factor in several physiological and pathological pathways, and it is synthesized by the nitric oxide synthase (NOS) . In invertebrates, including ticks, NO is related with a cytotoxic action against pathogens from hemocytes, derivates to phagocyte process during microbial infection . Now, three NOS isoforms have been described: the classic isoform inducible nitric oxide synthase (iNOS), the endothelial isoform (eNOS), and the neuronal isoform (nNOS) [91, 93]. Currently, the gene that codified for NOS has been identified and cloned from the insects:
5.2. Oxidative stress and detoxifying protein
In hematophagous arthropods, blood ingestion is the determinant of survival. However, during feeding and digestion, several toxic molecules are produced, such as reactive nitrogen species (RNOS) and reactive oxygen species (ROS) . The protection against nitrosative and oxidative stress is carried out by detoxification agents, produced largely by the midgut epithelial cells. In many insects, enzymes such as peroxiredoxins, catalases, and many members of antioxidant peroxidase family function as antioxidant agents. However, in arthropods, as in many organisms, the microbial infections are capable to induce oxidative stress. Suppression of pathogen ROS and RNOS induction in midgut facilitates the infection and microbial tissue dispersion . Interestingly, many arthropods have the capacity of enhancing ROS and RNOS against pathogen infection while simultaneously protecting their tissue cells with antioxidants. In this regard, the oxidize enzyme nicotinamide adenide dinucleotide phosphate (NADPH) of
5.3. Phenol oxidase and melanization
In arthropods, mechanical injury or the presence of foreign objects including pathogens results in melanin deposition around the damaged tissue or around the foreign object that in turn forms a capsule isolating the foreign particle. Melanins are molecules produced in the hemolymph by different types of hemocytes. The key enzyme for the melanization process is the phenol oxidase (PO). The metabolic pathway is initiated by hydroxylation of phenylalanine to tyrosine, followed by a series of reactions, resulting in 5,6-indolquinones, synthesized to phenol quinones, and these quinones polymerize to form melanin. The production of melanin is noticed by a dark and/or blackened color in the arthropod [110, 111, 112]. The signaling pathway starts with a hemocyte prophenol oxidase enzyme (PPO) synthesis (PO inactive form) that results in the conversion of the PPO into the active form by serine protease cascade . This molecular system is capable to recognize picomolar of bacterial lipopolysaccharide (LPS), peptide glycans, and fungi β-1,3-glucane. The intermediary components of this pathway, such as semiquinones, ROS, and melanin, are all very toxic to pathogens . On the other hand, the PPO-PO pathway in tick is little known. However, at the present, some studies in
6. Molecular approaches to tick immunology
6.1. Regulation of innate immune system in ticks
The innate immune systems represent one aspect in a generalized response to several pathogens and are composed of individual factors. This variability has a particular behavior in each tick. The principal components are the hemolymph and hemocytes; however, they are not the only factors. The response depends on the pathogen type, tissue, sex, life cycle phases, and tick species, among others. In this regard, innate immunity starts when membrane receptors recognize component characteristics of bacterial cell surfaces as peptidoglycans or lipoteichoic acid, which leads to synthesis of antimicrobial peptides (AMPs) as defensins, cecropins, attacins, and lysozyme that disrupt the cell wall structure, leading to cell death . Other components in the fungi cell wall are beta-1-3-glucans and beta-1-3 mannose or 2-keto-3-deoxyoctonate LPS, characteristic of Gram-negative bacteria, leading to soluble lectin synthesis . These cell wall components and foreign molecular structures are known as pathogen-associated molecular patterns (PAMPs) [2, 115]. In
6.2. Advance in molecular, functional genomics and proteomics in tick-host-pathogen interaction
Advances in gene identification and expression in tick tissues are being achieved by the use of expressed sequence tag (EST). The EST analyses correspond to partial sequence of acid nucleic from different random clones included in a cDNA library, obtained from the interest tissue mRNA . The analyses include the translation of EST sequence to amino acid sequence and compared with a public genome database. Interestingly, salivary gland genes of ticks show differential expression during blood ingestion, suggesting that processes are involved in homeostasis, tissue remodeling, immune defenses, angiogenesis, and the facilitation of the transmissible pathogen establishment . The EST library from unfed hard tick larvae of
7. Future directions
The ever-increasing knowledge of the immune system biology of vertebrates represents an important foundation in the research and development of advanced vaccines, new drugs, as well as the search for new targets for chemical or drug treatments of infectious diseases, which have contributed to the control of several human and livestock pathogens. Unfortunately, the immune system of invertebrates, especially, arthropod vectors like ticks, and their relationship with their pathogens, and infectious diseases they transmit, have been little explored. In this regard, the knowledge of mechanisms, molecules, and cells, as well as the regulation of immune response signaling pathways, represents an advance in designing control strategies that will contribute to improve livestock production and animal health. Currently, studies in insects and the molecular tool development help us to advance in the research to arthropod immune system regulation; however, there are many knowledge gaps about the ticks’ immune response. Elucidation of the different molecular pathways and their regulation in ticks’ immunobiology brings us closer to understand the role in the transmission of various infectious agents. Now, all transcriptome analyses and whole-genome sequencing represent powerful methodologies for understanding the biology, evolutionary relationships, and host-vector-pathogen interaction. The use of DNA/RNA sequencing modern tools could potentiate the discovery of different aspects that remain unsolved in tick biology, for the elucidation of the paradigms that currently remain unknown.
Hugo Aguilar-Díaz acknowledges the support given by Programa de Retenciones, Consejo Nacional de Ciencia y Tecnología (CONACYT), No. MOD- ORD-27 PCI-187-11-15.