Ticks (Acari: Ixodida) are ectoparasitic arthropods that obligatorily feed on the blood of a diverse list of vertebrate hosts, including mammals, birds, reptiles, and even amphibians. More than 950 tick species have been described to date, which, according to morphological and physiological characteristics, are divided into two main families, Ixodidae (hard ticks), comprising more than 75% of tick species, and Argasidae (soft ticks); a third family, known as Nuttalliellidae, is monospecific (1, 2). As a result of blood spoliation [a single ixodid adult female can ingest more than ~1 mL of blood (3)], the host can suffer from anemia, which negatively impacts the productivity of livestock and causes a huge economic burden worldwide. For example, the estimated annual losses due to reductions in weight gain and milk production caused by the cattle tick Rhipicephalus microplus are approximately 3.24 billion dollars in Brazil alone (4).

In addition to ingesting blood, ticks also secrete saliva into the host during feeding. Tick saliva, produced by their salivary glands, returns excess water and ions to the host, thereby concentrating the blood meal (5). Tick saliva contains an arsenal of bioactive molecules that modulate host hemostasis and immune reactions, thus enabling blood acquisition (6, 7). The antihemostatic and immunomodulatory properties of saliva can also facilitate the infection of pathogens that use saliva as a vehicle to be transmitted to the host during tick blood feeding (6, 8). Indeed, ticks are versatile vectors of viruses, bacteria, protozoans and nematodes, which cause life-threatening diseases to humans as well as to other animals, including livestock, pets, and wildlife (9). Among human diseases, we highlight Lyme disease, the most common tick-borne zoonosis, which is caused by spirochetes from the Borrelia burgdorferi sensu lato complex. After transmission by the bite of an infected tick, the typical clinical sign of Lyme disease is erythema migrans, but infection can spread and affect joints, heart, and the nervous system (10).

The first organ that a pathogen acquired within the blood meal interacts with is the tick gut (Figure 1). Then, the pathogen must colonize the gut epithelial cells and/or cross the gut epithelium to enter the hemocoel, an open body cavity filled with hemolymph, the fluid that irrigates all the tissues and organs in the tick. The pathogen must then reach the salivary glands. In each of these organs, the pathogen must counteract tick immune factors to be successfully transmitted through saliva to the vertebrate host in a subsequent blood-feeding (11). Some pathogens also have the ability to invade tick ovaries and can therefore be transovarially transmitted to progeny (Figure 1). Thus, elucidation of the immune factors involved in the interactions between ticks and tick-borne pathogens (TBPs) in each of these steps is essential to understand the biology of tick-transmitted diseases and may help to identify targets for the development of new strategies to block pathogen transmission. In this review, we present an update on humoral and cellular tick immunity components (Figure 1), including signaling pathways, antimicrobial peptides (AMPs), redox metabolism, complement-like proteins, and regulated cell death. Using a comparative approach with the immune system of other invertebrates, we highlight the challenges of studying tick immunity, the gaps, such as prophenoloxidase (PPO) and coagulation cascades, and the interconnections, such as immune system signaling pathway crosstalk. In addition, the role of tick microbiota in vector competence is also discussed.

The first records of studies on arthropod disease date to the 19^th century, when Louis Pasteur investigated the cause of brown dots on the cuticle of larvae of Bombyx mori that predestined larvae to death and affected silk production in France (12). In the 1980s, the isolation of several immune factors from the hemolymph of arthropods that have a large volume of hemolymph, such as larvae of dipteran and lepidopteran insects, horseshoe crabs and crayfish, was achieved. Indeed, the first animal AMP to be characterized was cecropin, isolated from the hemolymph of the moth Hyalophora cecropia (13). After that, AMPs were identified as important effectors of mammalian immunity (14, 15). In addition to AMPs, components of the PPO cascade from the hemolymph of H. cecropia (16), B. mori (17), and the crayfish Pacifastacus leniusculus (18) were also elucidated. Some years later, the components of the coagulation cascade, another important arthropod immune reaction, were characterized in P. leniusculus (19) and horseshoe crabs (20).

In the 1990s, relevant studies on the immune pathways that regulate AMP production were conducted using the fruit fly Drosophila melanogaster (hereafter referred to as Drosophila) as a model (21–24). Among them, we highlight the identification of a kappa B (κB)-binding region in the promotor region of certain insect AMP genes (25) and the identification of Toll receptors, posteriorly identified to be homologous to interleukin-1 receptor of mammals (26). Some years later, with the improvement of molecular techniques and funding by major support agencies, such as the MacArthur Foundation, the World Health Organization, and the National Institutes of Health (USA), studies on the arthropod immune system were redirected to vectors of human diseases, principally mosquitoes (27). In this period, Sanger-based technology was largely used to elucidate genomes and generate datasets of expressed sequence tags (ESTs). After the development of next-generation sequencing (NGS) technologies, additional information on arthropod genomes and transcriptomes was added to public databases (28). Indeed, currently, more than 40 arthropod genomes are available in the VectorBase database (https://www.vectorbase.org/organisms).

Knowledge of vector genomes and ESTs allowed in silico comparisons of immune factors among species [for example, see (29–33)]. Moreover, studies with diverse approaches, such as transcriptomics, proteomics, and metabolomics analyses, to assess the arthropod response to different microbial stimuli were significantly expanded in the postgenomic era (28). The development and application of RNA interference (RNAi) and CRISPR-Cas9 technologies to arthropods [(34, 35), respectively] were also important to determine the role played by immune factors in the interaction between vectors and vector-borne pathogens.

Despite the importance of ticks as disease vectors, studies on their genomes and the molecular factors involved in their interactions with pathogens are scarce compared to studies on other arthropod vectors. The large size of tick genomes and the high contents of repetitive regions make genome assembly difficult. Indeed, the size of tick genomes is approximately 1.3 Gbp in argasids and 2.6 Gbp in ixodids (36). However, the genome of the cattle tick R. microplus is even larger and has been estimated to be approximately 7.1 Gpb, which is more than twice the size of the human genome (37). In addition, approximately 70% of the tick genome includes repetitive regions (37, 38). For this reason, until very recently, only the genome of the tick Ixodes scapularis had been annotated (38). Additional genomes were recently assembled by the use of NGS (37, 39). The scarcity of studies on the molecular factors involved in ticks and TBPs is in part due to the need for sophisticated structures to raise vertebrate animals to feed ticks, which is laborious and involves ethical concerns. In the last few years, artificial feeding systems have been successfully used to maintain laboratory tick colonies; however, an animal blood source is still required (40). Finally, the development of continuous cell lines derived from tick embryos, despite representing a mixture of different cell types, has also contributed considerably to studies on tick biology and their interactions with TBPs (41).

Blood feeding represents a challenge for hematophagous arthropods due to the large diversity of pathogens to which these animals are exposed. In contrast to other arthropods, hard ticks are strictly hematophagous, feeding on the blood of their host for several days. In addition, some species feed on a different host in each developmental stage (larvae, nymphs, and adults), thereby increasing the chance of either acquiring or transmitting pathogens. Therefore, ticks are important vectors of a large list of disease-causing pathogens (42). In addition to host pathogens, ticks are in close contact with the microbiota of the host skin, which may also be acquired within the blood meal (43). Ticks are also exposed to microorganisms in the environment during the nonparasitic phases of their life cycle. Hence, the immune system of ticks must be activated continuously to protect them from harmful infections.

Most of our knowledge on arthropod immune responses has come from studies on dipteran insects, especially Drosophila and the mosquitoes Aedes spp. and Anopheles spp. In Drosophila, invading microorganisms are mainly recognized by the Toll, immune deficiency (IMD), Jun-N-terminal kinase (JNK), Janus kinase/signal transducer and activator of transcription (JAK/STAT) and/or RNAi pathways (44). Nonetheless, the hypothesis that the level of conservation of arthropod immune responses might be high has been rejected by several studies on ticks, mites, lice, hemipterans, and others, and it is now recognized that the immune system displays remarkable diversification across the Arthropoda phylum (30). Tick immunity is, however, still greatly neglected and unexplored (45). Hence, we review the prevailing knowledge on tick immune signaling pathways alongside the connections between them and other equally important factors, such as AMPs, redox metabolism, complement-like proteins, and regulated cell death.

AMPs are important effectors of the immune systems of both invertebrates and vertebrates, having a broad spectrum of activity against microorganisms (81). In ticks, the main sites of AMP expression are hemocytes, fat body, gut, ovaries, and salivary glands, where they can be modulated in response to either blood feeding or microbial challenge (82). Several reviews of tick AMPs addressing their characterization, as well as their interaction with microorganisms, have been published in the last decade (11, 33, 83, 84).

Interestingly, ticks use host hemoglobin, one of the most abundant proteins within the blood meal, as a source for the production of antimicrobial-derived fragments (85–88). Hemoglobin-derived AMPs, referred to as hemocidins (89), are produced by the proteolytic activity of aspartic and cysteine (catepsin-L like) proteinases from the tick gut (90). Structural studies with the synthetic amidated hemocidin Hb33-61a of R. microplus showed that its α-helical C-terminus is responsible for the permeabilization of the microbial membrane (91). However, it is still unknown whether hemocidins act intracellularly or if they are released to the tick gut lumen, where they can fight against microorganisms.

In addition to hemocidins, ticks also produce endogenous (ribosomally synthesized) AMPs (11, 83). Among the several tick AMPs identified to date, microplusins (also known as hebraeins) are among the most well characterized. Microplusin is a cysteine- and histidine-rich AMP that was first isolated from the hemolymph of adult R. microplus (92) and Amblyomma hebraeum (93). Microplusin was also identified in the ovaries and eggs of R. microplus (94), suggesting that in addition to protecting adults, it may also play a role in the protection of embryos before and after oviposition. Microplusin exhibits an α-helical globular domain and chelates metal ions (95). The bacteriostatic activity of microplusin against the Gram-positive bacterium M. luteus was reversed by the addition of copper II but not iron II. Indeed, microplusin interferes with the respiration (a copper-dependent process) of both M. luteus (95) and the fungus Cryptococcus neoformans (96). Microplusin was also reported to affect melanization and capsule formation, which are important virulence factors of C. neoformans (96). Interestingly, knockdown of microplusin increased the load of R. rickettsii in Amblyomma aureolatum (97). On the other hand, this AMP had no effect on either rickettsial transmission or tick fitness. Defensins compose another class of AMPs that have been described in several tick species, displaying activity against different types of microorganisms [for review, see (11, 83)]. For example, defensin-2 of Dermacentor variabilis was shown to protect against another bacterium of the genus Rickettsia, R. montanensis, as its neutralization with antidefensin-2 IgG increased the rickettisal load in the tick gut (98). Defensin-2 causes permeabilization of the bacterial membrane with consequent leakage of cytoplasmic proteins (98).

Dae2 is an AMP of I. scapularis that was acquired by horizontal bacterial gene transfer and has become an important effector to control B. burgdorferi infection (99), although it does not exhibit direct action on this pathogen (63). Indeed, it was recently shown that Dae2 is physically unable to overcome the outer membrane structure of the outer membrane of Gram-negative bacteria; thus, it does not present lytic activity against B. burgdorferi, suggesting the need for other factors, such as membrane-permeabilizing agents (100). As Dae2 is delivered to the vertebrate host bite site via saliva and exhibits strong activity against bacteria usually encountered in the host skin, this AMP may protect ticks from the acquisition and proliferation of host skin microbes (100).

Serine proteinase inhibitors have also been reported to play a role in the arthropod immune system. For instance, serine proteinase inhibitors mediate both coagulation and melanization processes of hemolymph and the production of AMPs (101). In addition, serine proteinases may also exert antimicrobial activity, possibly inhibiting proteinases that microorganisms use to colonize host tissues and evade the immune system (102). The first report of a tick serine proteinase inhibitor with antimicrobial properties was the ixodidin of R. microplus (103), which presents the key features of trypsin inhibitor-like domain proteins (104). Interestingly, one Kunitz inhibitor was reported to control R. montanensis infection in the gut of D. variabilis (105). In contrast to defensin, D. variabilis Kunitz-type inhibitors present a bacteriostatic effect on R. montanensis (106). Therefore, serine proteinase inhibitors are also used by ticks as powerful antimicrobial molecules.

Despite the diverse nature of molecules used by ticks as antimicrobials, little information on their synthesis regulation is available, as discussed above. Therefore, additional studies on the regulation of tick AMPs by immune signaling pathways are required to better understand their role in the control of distinct pathogens.

In addition to AMPs, triggering of the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in response to infection has been described in several arthropods, such as Drosophila (107) and mosquitoes (108). ROS have an essential role in infection-related physiological as well as pathophysiological processes, such as signaling, regulation of tissue injury and inflammation, cell survival, proliferation, differentiation, and apoptosis (109, 110).

In ticks, there is still little available information on ROS metabolism and their impact on pathogen control. Nonetheless, it is recognized that hemocytes produce ROS under stimulation. Gram-positive bacteria, zymosan, and phorbol 12-myristate 13-acetate elicit the production of hydrogen peroxide (H₂O₂) and superoxide ( O 2 − ) by hemocytes of R. microplus (111). In contrast, stimulation with lipopolysaccharide (LPS), the major component of the Gram-negative bacterial outer membrane, failed to induce ROS generation, indicating that different mechanisms or roles for ROS upon infection with either Gram-positive or Gram-negative bacteria may exist (111). Further studies with R. microplus showed that cytochrome c oxidase subunit III (COXIII), an enzyme of mitochondrial electron transport complex IV involved in mitochondrial ATP and ROS generation, is important for the transmission of A. marginale to calves (112). It is possible that COXIII knockdown imbalances tick redox metabolism, affecting its ability to transmit this pathogen (112). The peroxiredoxin Salp25D from I. scapularis had no effect on the transmission of B. burgdorferi but instead played a role in spirochete acquisition by the tick (113). RNAi-mediated silencing of Salp25D affects bacterial acquisition by ticks fed on B. burgdorferi-infected mice. The same effect was obtained when ticks were fed on Salp25-immunized mice (113). It is possible that Salp25 may detoxify ROS at the tick feeding site and gut, thus affording a survival advantage to B. burgdorferi.

In the mosquito An. gambiae, an extracellular matrix crosslinked by dityrosine covalent bonds catalyzed by dual oxidase (DUOX) and heme peroxidase is located in the gut ectoperitrophic space (between the epithelial cell layer and the peritrophic matrix). This extracellular matrix acts as an additional physical barrier to decrease gut permeability to bacterial PAMPs, impairing immune response activation by the resident microbiota (114). Importantly, the dityrosine network also provides a favorable environment for Plasmodium development, as it prevents the activation of nitric oxidase synthase (iNOS), a nitric oxide-generator enzyme (114). iNOS is responsible for parasite nitration, a key step in the action of the antiplasmodium complement-like molecule TEP1. Later, it was shown that the heme peroxidase 2/NADPH oxidase 5 system plays a central role in epithelium nitration, therefore potentiating the antiparasitic effect of nitric oxide (115). Similar to An. gambiae (114), an extracellular matrix was described in the tick I. scapularis, which acts as a shield that favors B. burgdorferi survival and indirectly prevents the induction of borreliacidal agents in the tick gut (116).

Intriguingly, A. marginale upregulated the genes encoding antioxidant enzymes, including superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase, thioredoxin, thioredoxin reductase, and peroxiredoxin, whereas genes encoding ROS-generating enzymes, such as DUOX and endoplasmic reticulum oxidase, were downregulated in R. microplus-derived BME26 cells (117). Conversely, R. rickettsii and heat-killed S. cerevisiae, E. cloacae or M. luteus triggered the opposite gene expression pattern (117). Furthermore, simultaneous RNAi knockdown of catalase, thioredoxin, and glutathione peroxidase, three representative members of the tick antioxidant enzymatic system, as well as the oxidation resistance 1 (OXR1), which regulates the expression of ROS detoxification enzymes, decreased A. marginale infection (117). Therefore, while BME26 cells respond to infection, producing an oxidant environment, A. marginale seems to subvert this response to create an antioxidant environment, which is required for its survival (117). It is possible that A. marginale manipulates R. microplus redox metabolism (and production of immune signaling pathway effectors, as aforementioned) to favor its proliferation. Additional studies are required to elucidate the mechanisms that this bacterium uses to subvert tick immune responses.

Hemocytes, which are sessile or circulating cells from arthropod hemolymph, are responsible for several immune responses. The nomenclature of hemocytes varies considerably depending on the arthropod species and/or the approaches of the study (118). Earlier morphological, ultrastructural, and physiological studies of the hemocyte repertoire in different tick species consistently reported the presence of three basic types of hemocytes, namely, phagocytic plasmatocytes and granulocytes and nonphagocytic granulocytes (119–121). These cells apparently differentiate from rarely occurring prohemocytes (120, 122). More recent studies have described additional types of tick hemocytes, namely, adipohemocytes in Rhipicephalus sanguineus (123) and spherulocytes and oenocytoids in R. microplus (122). The most important immune responses of arthropod hemocytes are phagocytosis, encapsulation, nodulation (which involves melanization by the PPO cascade), coagulation, and production of immune-related molecules.

The role of tick hemocytes in the phagocytosis of a variety of microbes, including bacteria, yeast, spirochetes, and foreign particles, has been investigated by several studies [for example, see (111, 124–127)]. By contrast, very little is known about the encapsulation and nodulation mechanisms. Indeed, there is only one report on encapsulation (128) and one on nodulation (129), both in D. variabilis. After the inoculation of ticks with Escherichia coli, hemocytes did not form circular layers but aggregated around the bacteria, which is a characteristic feature of nodule formation (129). As the encapsulation study was performed using an implant of Epon−Araldite under the tick cuticle, it is still unknown whether it also occurs against microorganisms (128).

In invertebrates such as insects and crustaceans, hemocytes produce components of the melanization response, which involves an enzymatic cascade referred to as the PPO activating system, ultimately resulting in the production of melanin (130, 131). This process can be locally activated by cuticle injury or systemically triggered by microbial invasion of the hemocoel. Interestingly, in the cotton bollworm Helicoverpa armigera, infection with the baculovirus HearNPV decreased the levels of the majority of PPO cascade components, while serpin-9 and serpin-5 (which were also shown to regulate the proteases cSP4 and cSP6, respectively) were increased (132). In addition, in vitro assays showed that hemolymph melanization can kill baculovirus, an effect abolished by the specific PO inhibitor phenylthiourea. Together, the results suggest that baculovirus inhibits the melanization response to ensure its survival in H. armigera (132). There is no evidence of the existence of the PPO cascade in ticks based on available genomic and transcriptomic data. In line with this, no PPO activity has been reported to be present in the hemolymph of the hard ticks Amblyomma americanum, D. variabilis, and I. scapularis (133). In contrast, two studies reported PPO-like activity using L-DOPA as a substrate in the hard tick R. sanguineus (134) and in the soft tick Ornithodoros moubata (135). However, the enzymes responsible for such activity have not yet been identified, and enzymatic assays did not employ phenylthiourea as a control.

Coagulation is another important immune response of arthropods. The final product of coagulation is a protein clot, which is essential to avoid the loss of hemolymph in cases of an injury and the spread of an invader microorganism throughout the hemocoel (136). In horseshoe crabs, the clotting process involves a serine-protease cascade that leads to the activation of the clotting enzyme that converts the coagulogen into the insoluble clot (137), while in crayfish, the process depends on direct transglutaminase (TG)-mediated cross-linking of a specific plasma protein homologous to vitellogenins (19, 136). TG is also involved in the final step of coagulation in horseshoe crabs, cross-linking coagulin with hemocyte surface proteins named proxins (138). Interestingly, factors of the coagulation cascade interact with hemocyanin, causing it to present PO activity in the horseshoe crab Tachypleus tridentatus, demonstrating crosstalk between melanization and coagulation cascades (139). In Drosophila, coagulation and PO activity were also described to be tightly associated (140). Wound sealing in flies involves two steps: in the first step, TG-mediated crosslinking of hemolymph proteins occurs, and in the second step, PO-dependent crosslinking takes place, hardening the clot and producing melanin. In ticks, putative coagulation was uniquely reported for D. variabilis, where a fibrous matrix was observed around an inert implant (128). TGs and proclotting enzyme precursors have been detected in tick genomes (33). Moreover, an injury-responsive multidomain serine protease homologous to Limulus Factor C has been characterized in I. ricinus (141). Therefore, additional studies based on appropriate in vitro assays are needed to ultimately resolve the question of the existence of hemolymph clotting in ticks.

Tick hemocytes (83), as well as the hemocytes of other arthropods, such as mosquitoes (142), also produce a series of immune-related molecules. Intriguingly, the hemocytome of I. ricinus showed that only 1.48% of the 15,716 coding sequences (CDSs) identified were related to immune factors (143). Of the identified CDSs, 327 were five times more highly expressed in hemocytes than in salivary glands and the gut, among which 11 encode immune factors, including AMPs and proteins involved in pathogen recognition. As presented in this section, hemocytes are versatile components of the arthropod immune system that play diverse and key roles. The principal insect tissue that produces the majority of soluble immune molecules in hemolymph is the fat body (44). The role of tick fat body in the tick immune system requires further investigation.

One important branch of both cellular and humoral innate immunity in vertebrate and invertebrate metazoan organisms is carried out by the complement system. In higher vertebrates, the complement system is composed of approximately thirty components arranged in classical, lectin, and alternative pathways, which recognize foreign cells (microbes), specifically tag them via opsonization, and ultimately, eliminate them via phagocytosis or cell lysis (144). The common denominator of all three pathways is the proteolytic activation of the central C3 complement component. The occurrence of this molecule can be traced back in most ancient invertebrates, such as horseshoe crabs (subphylum Chelicerata, class Merostomata), implying that an ancestor of the complement system existed on Earth for more than 500 mil. years (145, 146). For ticks, which are also chelicerates, advanced knowledge of the primitive complement system of horseshoe crabs gathered during the past two decades presents the best matching comparative model (137, 145, 147).

Microbial pattern recognition by the vertebrate lectin pathway is mediated by multimeric mannose-binding lectins (MBLs) or ficolins. The horseshoe crab counterparts of mammalian ficolins are lectins named tachylectin-5 or carcinolectin-5 (148–150). These lectins share a fibrinogen-related protein (FRED) with ficolins but lack the N-terminal collagen-like domain responsible for forming complexes with MBL-associated serine proteases (MASPs) (151), which are absent in arthropods (146). The lectin Dorin M, purified from the plasma of the soft tick O. moubata (152), was shown to be a clear ortholog of the horseshoe crab tachylectins-5 (153), and similarly to ficolins and tachylectins, it forms high molecular weight multimers in the native state (152). The search for homologous lectins in I. ricinus (154) and in the genome of I. scapularis (155) revealed the existence of two phylogenetically distinct families, further referred to as ixoderin A and ixoderin B (Figure 4). Ixoderin A is mainly present in plasma and is responsible for the hemagglutination of mouse erythrocytes (155). On the other hand, the salivary gland transcriptomes of I. ricinus (157, 158) indicate that ixoderin B represents a highly variable multigene family that is preferentially expressed in the salivary glands and secreted into saliva. The function of these FREPs in tick saliva is still obscure, but we can hypothesize that they may play a role in the recognition of a specific tick host.

The central effector molecules of vertebrate and invertebrate complement systems are proteins belonging to the thioester-containing protein (TEP) family, formerly referred to as proteins of the α₂-macroglobulin superfamily (144, 159, 160). The TEP designation is given due to the presence of a highly reactive β-cysteinyl-γ-glutamyl thioester (TE) bond within a thioester domain. Invertebrate TEPs are divided into four major phylogenetically distinct groups: (i) panprotease inhibitors of the α₂-macroglobulin type (α₂M), (ii) C3-like complement components (C3), (iii) insect-type TEPs (iTEPs), and (iv) macroglobulin complement-related proteins (MCRs) (124, 146, 161). Genome-wide screening of the I. scapularis genome (124) and the recently available horseshoe crab genomes (162, 163), together with transcriptome data from a variety of arthropods, reveal that all these major groups of TEPs are present in chelicerates, but C3-like molecules are absent in crustaceans and hexapods, while α₂Ms were lost in the evolution of some insect lineages, such as Drosophila and mosquitoes (146).

Orthologs of nine TEPs present in the I. scapularis genome (124) were identified in closely related I. ricinus (125), and their full CDSs were recently deposited in GenBank: IrA2M-1 (MT779788); IrA2M-2 (MT779789); IrA2M-3 (MT779790); IrTep (MT779791); IrC3-1 (MT779792); IrC3-2 (MT779793); IrC3-3 (MT779793); IrMcr-1 (MT779795); and IrMcr-2 (MT779796). The domain structure of tick TEP representatives is shown in Figure 4. The hallmark domain of α₂Ms is the presence of the bait region (BR), which is cleaved by the target protease. Several bait region alternative splicing variants were reported in the α₂M region of the soft tick O. moubata (164) as well as in IrA2M-1 of I. ricinus (165). IrTEP has a domain architecture quite similar to that of IrA2Ms; however, this molecule is phylogenetically more closely related to insect TEPs (124). Tick C3-like molecules (IrC3-1, IrC3-2, IrC3-3) possess two signature domains, namely, the anaphylatoxin domain and C-terminal NTR complement_C345C domain. The MCRs can be clearly identified based on the presence of the short low-density lipoprotein receptor domain (LD), which occurs in the central part of the molecule (Figure 4). Tick TEPs are specifically expressed in tick fat body (IrA2M-1, IrA2M-3, IrC3-1, IrC3-2, IrC3-3), tick hemocytes (IrA2M-2, IrA2M-3), salivary glands (IrC3-2, IrMcr-1), and ovaries (IrTEP) (125).

Other characterized components of the I. ricinus primitive complement system are two putative convertases: (i) IrC2/Bf (156), which is related to the vertebrate complement components C2 and/or FactorB (Bf) (144) and homologous to convertases from horseshoe crabs (145, 166), and (ii) IrFC (141), homologous to Limulus Factor C, which plays a dual function as the factor that triggers the clotting cascade upon sensing Gram-negative bacterial endotoxins and as an LPS-sensitive convertase of the horseshoe crab C3 complement component (147, 167). Both IrC2/Bf and IrFC are multidomain convertases that share the N-terminal trypsin-like domain and numerous CCP modules (complement control protein, aka sushi domains) (Figure 4). While IrC2/Bf is mainly expressed in the tick fat body and its expression is responsive to injection of the yeast Candida albicans and a variety of Borrelia species (156), IrFC is produced by tick hemocytes, and its expression is responsive to any injury, including injection of sterile phosphate-buffered saline, implicating its role in hemolymph clotting and wound healing (141).

RNAi-based functional studies of I. ricinus complement components successively deciphered their nonredundant roles in the phagocytosis of different microbes by tick hemocytes (124, 125, 141, 155, 156, 165). Phagocytosis of Gram-negative bacteria represented by the tick pathogen Chryseobacterium indologenes (168) depends mainly on the convertase IrFC, which seems to be linked to the IrC3-3 component. Interestingly, phagocytosis of this bacterium is also clearly mediated by α₂Ms IrAM2-1 and IrAM2-2 by a yet unknown mechanism that likely involves the interaction of these macromolecular protease inhibitors with the potent metalloprotease secreted by the bacterium (168).

A distinct phagocytic pathway dependent on the convertase IrC2/Bf is responsible for the phagocytosis of the yeast C. albicans and spirochete Borrelia. Phagocytosis of C. albicans is further facilitated by IrC3-1 and IrMcr-2, consistent with the reported role of its related molecule MCR (DmTep6) in the phagocytosis of this yeast by Drosophila S2 cells (161). Similar to other Gram-negative bacteria, phagocytosis of Borrelia is also mediated by IrC3-3. Ixoderins A and B were found to be involved in the phagocytosis of all the tested microbes, except Borrelia. Although Borrelia afzelii (the principal Lyme disease-causative agent in Europe) is actively phagocytosed by tick hemocytes; neither RNAi-mediated silencing of any tick complement-related molecules nor the total elimination of phagocytosis by preinjection of latex beads have shown any effect on the transmission of these spirochetes to the host (126). These results indirectly support the recent finding that the transmission of B. afzelii from infected I. ricinus nymphs to naive mice avoids the tick hemocoel and salivary glands and occurs by a direct gut-to-mouthpart route (169). However, it is possible that the tick complement plays a role in the transmission of other tick-borne pathogens, such as intracellular bacteria, including Anaplasma spp. and Rickettsia spp., or protozoan parasites, including Babesia spp. These objectives await an intensive research focus in the future.

Regulated cell death (RCD) is widely distributed in nature, occurring in both unicellular and multicellular organisms (170). As extensively stated above, the arthropod innate immune system must coordinate pathogen recognition with effector mechanisms to successfully control infection. Nonetheless, over the past decade, several studies have established RCD processes as important mechanisms for the regulation of the immune response as well as the control of infections (171–173). Autophagy, apoptosis, and necrosis are the main types of RCD that have been described to be related to insect immunity in the last few years (173). In this section, we focus on autophagy and apoptosis and their interconnections with immune signaling pathways.

Autophagy is a highly conserved process in which endogenous material (misfolded proteins and aggregates, damaged organelles, and other macromolecules) or exogenous material (such as invading pathogens) are selectively recognized and sequestered within autophagosomes (double-membrane vesicles) that subsequently fuse with lysosomes, leading to cargo degradation (174). Autophagy is executed by a series of evolutionarily conserved autophagy-related (ATG) proteins that have orthologs in eukaryotic organisms ranging from yeasts to humans (174). Studies on Drosophila have provided excellent insights into the importance of autophagy during microbial infection (175). For instance, infection with the intracellular bacterium Listeria monocytogenes induces autophagy in both hemocytes and a hemocyte-derived Drosophila cell line (176). Interestingly, the IMD pathway receptor PGRP-LE is involved in bacterial recognition for autophagy activation. In addition, RNAi-mediated silencing of the autophagy genes atg5 and atg1 increases the bacterial load within cells, showing that this pathway is important to control infection. Autophagy is also important to control infection by vesicular stomatitis virus (VSV) in Drosophila (177, 178). In this process, the viral glycoprotein VSV-G is recognized by Toll-7, activating autophagy via a still unknown pathway that is independent of the canonical Toll, IMD, and JAK/STAT pathways (178). The role played by autophagy in the protection of mosquitoes against viruses is somewhat controversial, with reports suggesting both pro- and antiviral effects (179). In ticks, the expression of atg genes was upregulated under starvation in Haemaphysalis longicornis (180, 181), I. scapularis (182), R. microplus, and A. sculptum (183), correlating with the classical role of autophagy in stress. However, studies correlating tick autophagy with immune responses still need to be performed.

Apoptosis is another highly conserved RCD that is essential for removing damaged and infected cells to maintain homeostasis. There are two major apoptotic signaling pathways: extrinsic, also called death receptor pathway, and intrinsic, in which mitochondria play a central role (Figure 5). Both of these pathways culminate in the activation of executer caspases that are key for eliminating apoptotic cells (184). Apoptosis is activated in Drosophila by infection with Drosophila C virus (DCV), and infected cells are phagocytized by hemocytes in a phosphatidylserine-mediated process (185). Apoptosis can also control viral infection in mosquitoes (186, 187). Interestingly, the expression of proapoptotic genes was significantly higher in the refractory strain Cali-MIB of Ae. aegypti than in the susceptible strain Cali-S upon experimental infection with DENV-2, suggesting that apoptosis is involved in the distinct susceptibility of mosquitoes to infection (186). Apoptosis is also involved in the control of the proliferation of WNV in the midgut of a refractory strain of the mosquito Culex pipiens pipiens (187). Studies on the apoptotic response upon pathogen infection in ticks are also scarcer than those in insects. Infection of the I. ricinus cell line IRE/CTVM20 with the bacterium A. phagocytophilum and the flaviviruses TBEV and LIV upregulated the expression of apoptosis-associated components, such as cytochrome c and fatty-acid synthase (FAS) (54, 188).

To guarantee their replication and survival within the host cell, many pathogens, including viruses, bacteria and protozoa, subvert apoptosis induced by infection (189). For instance, infection with Zika virus (ZIKV) inhibits apoptosis in Ae. aegypti through the action of sfRNAs (190). Until very recently, the unique example of a pathogen that inhibits apoptosis in tick cells was A. phagocytophilum (188, 191, 192). This bacterium inhibits the intrinsic apoptosis pathway in I. scapularis salivary glands and ISE6 cells by porin (voltage-dependent anion-selective channel) downregulation, resulting in the inhibition of cytochrome c release. Nonetheless, while the intrinsic pathway is inhibited, the extrinsic pathway seems to be activated through the inhibition of FAS by an unknown mechanism as a possible attempt to limit bacterial infection (192, 193). Conversely, in the I. scapularis gut and I. ricinus IRE/CTVM20 cells, A. phagocytophilum supposedly inhibits apoptosis through upregulation of the JAK/STAT pathway (191). However, these conclusions were mostly based on transcriptomics and proteomics data, and only a few genes were functionally characterized by RNAi. In addition, the effectors that A. phagocytophilum uses to inhibit tick apoptosis have not been elucidated to date, as they have been for the manipulation of apoptosis in human neutrophils (194). Recently, it was reported that R. rickettsii downregulates negative regulators of apoptosis in the initial phase of BME26 cell infection, which are upregulated later. Infection also prevents the fragmentation of DNA and decreases the activity of caspase-3 as well as the exposure of phosphatidylserine. Remarkably, bacterial growth is higher in apoptosis-inhibited tick cells, suggesting that such an inhibitory effect is important to guarantee cell colonization (195).

Apoptosis is closely regulated by apoptosis inhibitor proteins (IAPs) (Figure 5) (196, 197). IAPs present at least two conserved motifs: baculoviral IAP repeat (BIR) motifs, which are represented from one to three tandem repeats in the N-terminus, and the C-terminal really interesting new gene (RING) motif; this last motif presents E3-ubiquitin ligase activity. In Drosophila, the E3-ubiquitin ligase activity of DIAP-2 has been described as being important for the activation of Relish after recognition of Gram-negative bacteria (198–201). Knockdown of the XIAP of I. scapularis from the IMD pathway, which also possesses E3-ubiquitin ligase activity, increased colonization by A. phagocytophilum, showing that E3 is important for the control of infection (202) (see the above section “The unconventional IMD pathway”). However, it is still unknown whether XIAP plays a role in tick apoptosis.

Additional studies are warranted to better understand the role played by tick apoptosis pathway components in infection control and their interconnections with immune signaling pathways as well as the mechanisms that pathogens use to subvert the death of tick cells, thereby guaranteeing their survival and proliferation.

Ticks, as well as most multicellular eukaryotes, possess associated bacteria, viruses, fungi and archaea, mainly in mucosal organs, composing their microbiota (203). In the last decade, several studies have focused on the bacterial composition of different genera of ticks and have explored the interaction of TBPs with nonpathogenic tick endosymbionts to elucidate the impact of microbiota on their vector competence (62, 64, 204–206). The tick immune responses to microbiota, despite its importance for a more comprehensive understanding of tick biology, is a field that requires attention since little is known in comparison with other arthropods. Therefore, in this section, we summarize the Ae. aegypti immune responses to the gut microbiota and relate this knowledge to ticks.

In adult mosquitoes, the IMD pathway is activated in response to microbiota proliferation induced by the blood meal, limiting Sindbis infection (207). ROS production is mediated by DUOX, whose expression is regulated by a gut membrane-associated protein named Mesh (208). However, a reduction in ROS due to heme release upon blood digestion protects the gut microbiota (209). To counteract the action of Relish-dependent AMPs, the gut microbiota stimulates the expression of C-type lectins (CLTs) in Ae. aegypti, which bind to bacterial cell walls, thereby protecting the bacteria (210).

In addition to the immune response to microbiota, there is interest in the impact of microbiota on the vector capacity of mosquitoes and ticks. In Ae. aegypti, several studies have shown that larval microbiota can influence vector competence in adult mosquitoes, playing a critical role in their response to viral infections (211). For instance, E. coli infection during the larval stage stimulates the production of AMPs and nitric oxide, protecting the mosquito from other infections (212). In addition, when Enterobacteriaceae bacteria are the only members of the larval microbiota, DENV infection in adults is reduced in comparison to Salmonella sp. as the only member (213). Conversely, exposure to pathogenic Bacillus thuringiensis subsp. israelensis in resistant larvae increases adult susceptibility to DENV [but not to Chikungunya (CHIKV)] (214), possibly due to changes in the microbial community (215).

Of all the bacteria present in insect microbiota, Wolbachia pipientis may be the most ubiquitous symbiont, as it is naturally present in 40% of all terrestrial arthropod species (216). Intracellular and maternally transmitted Wolbachia can cause pathogen interference (PI; the ability to reduce the chance of pathogen infection and decrease pathogen load) and cytoplasmic incompatibility (CI; when infected males mate with uninfected females, the hatch of eggs is heavily reduced), manipulating host reproduction and working as a genetic driver (the ability to spread through a population in a non-Mendelian way) (217). In Ae. aegypti, Wolbachia strongly reduces CHIKV, DENV and ZIKV infection and vector competence via the PI phenotype (217–220). For this reason, there is an ongoing program, the World Mosquito Program, to infect mosquito eggs with Wolbachia (from Drosophila – wMel) in the laboratory and release them in dengue-endemic areas, such as the city of Rio de Janeiro in Brazil (221, 222).

Despite being the most common bacteria in the microbiota of insects, Wolbachia has been reported only in a few species of ticks (205, 223, 224). In fact, the adult tick microbiota is mostly composed of Coxiella, Rickettsia, Francisella, Spiroplasma, Midichloria and Rickettsiella (203, 205). Most of these bacteria are intracellular and noncultivable in the laboratory, which hampers the manipulation of tick microbiota. Some studies on the larval microbiota of I. scapularis (62, 64, 204) showed a wide variety of bacterial genera, including cultivable extracellular bacteria. A deep bioinformatics analysis of the raw data of these studies suggested a taxonomic core composed of 61 bacterial taxa for I. scapularis larvae (225). However, this high number of bacterial genera in the larval microbiota of I. scapularis has been questioned, and the possibility of contamination potentially due to the low biomass of tick samples has been raised (226, 227). Interestingly, some tick species, such as R. microplus and I. ricinus, present a poor and unstable microbiota in the gut (228). On the other hand, these two species harbor a more abundant and stable microbiota in their ovaries that is composed mostly of Midichloria spp. in I. ricinus (228) and Coxiella spp. in R. microplus (229). The authors hypothesized that the reduced microbiota in the tick gut might be due to the action of immune factors, such as AMPs and ROS (228).

As described for mosquitoes, the tick microbiota can exert an effect on vector capacity; bacterial infection can modify the microbiota of its host. In I. scapularis, perturbation of the normal gut microbiota decreased the expression of STAT, which, in turn, reduced the expression of peritrophin-1. Since the integrity of the peritrophic matrix is essential to B. burgdorferi infection, as previously discussed, alteration of the microbiota reduces borrelial colonization (62). In addition, infection with B. burgdorferi promotes the expression of the I. scapularis gene pixr, which encodes a gut secreted protein with functions in tick biology, such as larval molting and inhibition of biofilm formation (preferentially by Gram-positive bacteria), facilitating the colonization of B. burgdorferi in ticks (204). These studies suggest a mutual influence or interconnection between the gut microbiota and B. burgdorferi in I. scapularis. Conversely, A. phagocytophilum infection in this same tick species promotes the expression of an antifreeze protein, which perturbs the gut microbiota and reduces the integrity of the peritrophic matrix (64). In contrast to B. burgdorferi (62), an extracellular bacterium that benefits from a preserved peritrophic matrix, A. phagocytophilum, which is an obligate intracellular bacterium, reduces the thickness of the peritrophic matrix to colonize the tick gut (64). In the tick Dermacentor andersoni, a microbiota alteration was induced by feeding on calves treated with oxytetracycline. Although this treatment did not change the microbiota composition, the proportion of its components was altered, negatively impacting the acquisition of A. marginale and Francisella novicida (230). Importantly, perturbation of the D. andersoni microbiota exerted a negative impact on the reproductive fitness of the tick, thereby identifying the microbiota as an important target for the development of control strategies (231).

A recent study compared the microbiota of two R. rickettsii tick vectors in Brazil, Amblyomma sculptum and A. aureolatum, which present significant differences regarding their susceptibility to infection (232). Interestingly, A. aureolatum is highly susceptible to R. rickettsii infection and harbors a robust intestinal microbiota, mainly composed of the Francisella genus. A. sculptum, on the other hand, is less susceptible to R. rickettsii infection and harbors a reduced intestinal microbiota (206). Additionally, R. rickettsii causes a slight reduction in the microbiota load without changing its composition. It has been reported that the transcriptional gut response of these two ticks to R. rickettsii infection is also distinct: while the majority of genes of A. sculptum, including immune factors, were upregulated by infection, A. aureolatum genes were mostly downregulated (233). Together, these data suggest that the A. aureolatum gut microbiota somehow desensitizes the immune system and promotes R. rickettsii infection. Interestingly, the presence of Francisella endosymbionts positively impacted the establishment of F. novicida in D. andersoni, and the authors hypothesized that these endosymbionts may suppress the tick immune system, favoring F. novicida acquisition (230). Additional studies are needed to confirm this hypothesis and to identify the mechanisms by which microbiota delineate tick susceptibility to infection.

Studies focused on the immune response to tick microbiota are necessary to elucidate this important feature, which is involved in many aspects of tick biology, including its vector capacity, representing a question of public health interest. These responses may involve only one immune signaling pathway or crosstalk of the different pathways, as is suggested to occur in R. microplus in response to A. marginale infection (49). In addition, we can raise a possible role of DUOX in the control of tick microbiota since this enzyme is present and functional in I. scapularis (116).

To date, studies on the interactions between ticks and TBPs have shown that both the IMD and JAK/STAT pathways are key for the control of bacterial infections (B. burgdorferi, A. marginale and A. phagocytophilum), while the Toll and RNAi pathways might be involved in tick defense against viral infections. Studies on the identification of tick immune system-specific effectors are warranted to describe the mechanisms involved in these signaling pathways. In addition, this review has provided several lines of evidence of interconnections between immune signaling pathways, as well as links among several elements from the innate immunity of arthropods, such as the RNAi system, redox metabolism and microbiota. This review also highlights the importance of bearing in mind a widely integrated, versatile, and complex immune system as a response to infection in ticks, far beyond a canonical and linear pathway.

As mentioned above, most of our knowledge on arthropod immunity comes from Drosophila as well as other arthropod studies. Nevertheless, the search for a direct correlation between immune signaling and effector specificity in ticks may result in the absence of new and important mechanisms of the tick immune system. For instance, recent works have suggested that ticks express different types of effectors from immune signaling pathway activation, such as peritrophins, which are key structural components of the gut peritrophic matrix. Moreover, it is extremely important to keep in mind that Drosophila is not a vector model and, as such, some aspects of the pathogen-vector interaction cannot be fully modeled. Of relevance, the studies on the innate responses of Drosophila follow infections by an intrathoracic injection with large loads of artificial pathogens, greatly contrasting those from ticks, which experience natural pathogens, doses, and routes of infection.

Phagocytosis, AMPs, complement-like molecules and ROS production are also considered important factors for protecting ticks from infection. The microbiota can interfere with tick colonization by pathogens as well. Therefore, it is important to identify the microorganisms that compose the microbiota of different organs of ticks and to determine their influence on the tick immune system as well as on tick vector competence. Interestingly, in some insects, the immune system can be primed by nonpathogenic microorganisms, protecting the animal from subsequent infection with a pathogenic microbe (234). Nonetheless, there is only one report on tick immune system priming to date (48). The authors showed that immune priming with POPG and PODAG protects I. scapularis against infection by A. phagocytophilum and D. andersoni against infection by A. marginale. In addition, it was shown that the lipid immune-priming effect is abolished only by the silencing of IMD pathway components but not of the Toll or JAK/STAT pathways, excluding an off-target effect (48). Therefore, the tick immune system and its relationship with microorganisms is a wide and unexplored field to be pursued.

In summary, every aspect concerning the tick immune system and its relation with microorganisms - endosymbionts or pathogens - remains far from completely understood. Despite the vast advances made in recent decades, which have helped us to build parts of this puzzle, working with ticks is still a bright and open field full of possibilities. We expect more groups to work with ticks due to their importance to public health and await the discovery of new knowledge in the next few years. In this way, we will all be able to assemble this extraordinary and complex puzzle.

All authors searched the literature and wrote the manuscript. SD and AF conceived the work and the final edition. GS, DP, EE, LM, and PK elaborated the figures. All authors contributed to the article and approved the submitted version.

This work was supported by funds from the São Paulo Research Foundation (FAPESP 2013/26450-2) and the National Council for Scientific and Technological Development (CNPq) - the National Institutes of Science and Technology Program in Molecular Entomology (INCTEM 573959/2008-0). AF (309733/2018-9) and SD (304382/2017-5) received CNPq research productivity scholarships. GS and DP received a postdoctoral scholarship from FAPESP (2019/07122-0 and 2018/00652-1, respectively) and EE from the Coordination for the Improvement of Higher Education Personnel (CAPES 88887.321638/2019-00). VU and PK were supported by the Czech Science Foundation (20-05736S).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.