Ticks: Ixodidae

Ticks are obligate blood-feeding ectoparasites and one of the most prominent arthropods harboring pathogens worldwide (De la Fuente et al., 2008;Jongejan and Uilenberg, 2004). In the United States, ticks are the number one arthropod vector of animal or human diseases (CDC, 2018;Dantas-Torres et al., 2012;Mullen and Durden, 2002;WHO, 2014;Sonenshine and Roe, 2013b). Ticks belong to the Order Metastigmata (Ixodida), which includes three families Ixodidae (hard ticks), Argasidae (soft ticks), and Nuttalliellidae, containing the species of human and veterinary importance. The family Ixodidae contains the main vector species, comprising more than 700 species (Guglielmone et al., 2010;Mullen and Durden, 2002;Sonenshine and Roe, 2013a). Ixodidae ticks are subdivided into two groups: Prostriata, containing one genus (Ixodes) and Metastriata containing 11 genera, which include Amblyomma, Dermacentor, Hemaphysalis, and Rhipicephalus (Sonenshine and Roe, 2013a).

All ticks are required to feed on a host in order to molt into the next stage (Sonenshine, 1991a), often allowing them to explore multiple hosts throughout their lifetime. Based on the lifestyle, ticks can be further divided into one-host, two-host or three-host ticks (Sonenshine and Roe, 2013a), although the majority of hard ticks fall under the three-host category. In the three-host life cycle, the ticks are required to feed on three different hosts (or three different attachments) in order to complete their life cycle and one single female can lay several thousands of eggs at once; the maximum record being Amblyomma variegatum with 36,206 eggs laid (Dipeolu and Ogunji, 1980). The life cycle of a hard tick can last from several months to multiple years including off-host state, during which their unique osmoregulatory physiology is required to survive under harsh environmental conditions. Hard ticks ingest a large amount of blood during a single blood meal, however it is estimated that they spend over 98% of their lifetime at off-host state (Sonenshine, 1991a;Sonenshine and Roe, 2013a). Therefore, tick survival is largely dependent on the environmental conditions, which include fluctuating temperatures and relative humidities (RH) (Needham and Teel, 1986). Consequently, tick success largely depends on their ability to maintain water balance in the environment with low RH (Needham and Teel, 1986;Norval, 1977).

Osmoregulation in Insects and Other Bloodfeeding Arthropods

Water is the basis of life as it makes up to 65 to 90% of all living organisms. All living organisms need to maintain the ideal concentration of ions or electrolytes within the cell for proper cellular functions. At the organismal level, gain of water requires uptake of dietary water while water loss occurs through the integument by evaporation and through the excretory system in the processes of eliminating metabolic wastes. Arthropods, including insects and arachnids, are especially susceptible to dehydration due to their small size and large surface-to-volume ratio compared to other animals. Their cuticle covered with a wax layer and other osmoregulatory mechanisms allow a large number of species withstanding the aridest environments (Berridge, 1970). When subjected to desiccation, the fruit fly (Drosophila melanogaster) can maintain a constant hemolymph osmolarity even after reducing their hemolymph volume down to below 25% (Albers and Bradley, 2004). Hematophagous arthropods, such as the kissing bug, the bed bug, fleas, and some ticks, have complicated ion regulation mechanisms to maintain water homeostasis during the blood meal (Benoit and Denlinger, 2010;Sonenshine, 1991b;Thiemann et al., 2003). Initial studies in the kissing bug (Rhodnius prolixus) and mosquitoes showed that the blood meal caused significant changes in the physiology of these arthropods. The blood meal provides an excess of Na⁺ and Cl^- ions, which need to be quickly eliminated from the body, leading to post-meal diuresis in blood-feeding insects (Coast, 2009;Orchard et al., 2021;Stobbart, 1977). In the case of ticks, the long duration of blood-feeding, up to more than a week of feeding, requires a more complex process including the gene regulations depending on the feeding phases. In addition, ticks rely heavily on their ability to absorb water from the environment without a nutritious diet during their long off-host periods, which indicates that an intricate process of tick water osmoregulatory is in place to survive prolonged periods of time without a blood meal.

Water Homeostasis in The Ixodid Tick

Ticks, like many arthropods, are at constant threat of evaporative loss of water due to their small size. Water loss through the integument is generally well prevented by the integument sclerotized with a cuticle rich in wax. However, other imminent water losses naturally also occur through hindgut excretion, salivation (Kim et al., 2016), and dermal secretion.

Water uptake

Ticks have the capability to uptake water from water vapor. Water vapor uptake directly depends on the Critical Equilibrium Activity (CEA), which is the minimum RH at which ticks can passively uptake water and maintain water homeostasis (Sauer and Hair, 1971;Sonenshine, 1991b;Wharton and Richards, 1978). In most ixodid ticks, the CEA is close to 90% RH (Wharton and Richards, 1978). In A. americanum, water vapor uptake occurs between 85% and 93% RH and accounts for up to 77% of the water uptake (Freda and Needham, 1984) in the experimental conditions. Water vapor uptake was first reported in A. variegatum with the mouthparts as the main route (Rudolph and Knulle, 1974). Ticks secrete hygroscopic saliva, during periods of dehydration, which is rich in chloride, potassium, and sodium ions (Fig. 1A) (Hsu and Sauer, 1974;Needham and Teel, 1986). This high ionic saliva then captures water vapor from the environment, and it is later ingested by the tick (Needham and Teel, 1986) (Fig. 1B). Although the ability of ticks to maintain water homeostasis has been of great interest in the last several decades, a better understanding of tick success in water regulation will potentially provide the tools for tick control.

It has been demonstrated that some species of ticks can actively drink water voluntarily to maintain water balance during off-host state. Water drinking behavior was observed for several Rhipicephalus species, D. nuttalli, A. hebraeum, and larvae of A. americanum (Londt and Whitehead, 1972;Splisteser and Tyron, 1986;Wilkinson, 1953). In contrast, some studies reported no water drinking behavior in Ixodes ricinus, D. marginatus, and D. reticulatus (Kahl and Alidousti, 1997;Lees, 1946;Meyer-Konig et al., 2001). While A. americanum and other metastriate ticks are generally directly drinking water (Fig. 1C) (Maldonado-Ruiz et al., 2020), prostriate ticks have a general tendency for avoidance to touch the water. However, it has been observed that a prostriate tick, Ixodes scapularis, approaches and touches water droplets and occasionally insert the hypostome (mouthparts) and drink water (5% of ticks tested) (Fig. 1D) (Kim et al., 2017). Other studies reported an increased weight gain in ticks, which was believed to occur not by the direct ingestion through the mouthpart (Knulle and Devine, 1972;Lees, 1948), but by being in physical contact with water droplets. These studies imply that further observations with varied conditions, such as the geographically different strains and varied environmental conditions, are required for a solid conclusion. Ingested water through the mouthparts is later observed in the internal tick organs, type I acini of salivary glands (Fig. 1E and 1F) and midgut, and transported into the hemolymph based on a study tracing the fluorescence dye in the ingested water (Kim et al., 2017). Tick-specific mechanisms of water uptake likely offer a new target system that could be utilized for the development of a novel tick control strategy. In addition, direct uptake of water in ticks is a potential route for delivering tick-specific toxic reagents, as we previously demonstrated (Maldonado-Ruiz et al., 2020). It has been shown that ticks are able to ingest different bacteria through voluntary drinking, and such bacteria can be later recovered from the tick gut. In addition, 100% tick mortality has been achieved in A. americanum, upon ingestion of phosphate-rich salt solutions by voluntary drinking. High levels of excretions through the hindgut of dying ticks were observed after ingestion of phosphate-rich salt solutions, suggesting the mortality was caused by excessive dehydration caused by the increased excretion. Nonetheless, 60% tick mortality was also achieved through voluntary drinking of water containing Pseudomonas aeruginosa, which is an opportunistic pathogen (Maldonado-Ruiz et al., 2020) in A. americanum. While it is unlikely that this bacterium can be used as a control strategy, this demonstrates mortality through voluntary ingestions in the unfed tick can be achieved and that this strategy can also be used for delivery of other tick-specific toxic compounds.

Main osmoregulatory organs and the control of water physiology

Excretory mechanisms in the ixodid ticks are similar to those observed in insects, but significantly different in the neural and hormonal components involved in the process (Sonenshine and Roe, 2013a). In hard ticks, the salivary glands (Fig. 1E, in blue), Malpighian tubules (Fig. 1E, in yellow), and hindgut (Fig. 1E, in green) are the main osmoregulatory organs. The excretory organs, hindgut and Malpighian tubules, are believed to be controlled by the brain of the tick (synganglion) (Fig. 1E, in pink) (Šimo and Park, 2014), while the mechanisms of tick salivary secretion are not yet fully understood (Kim et al., 2019).

Salivary glands

The salivary glands of ticks are the largest pair of glands in the Ixodid tick’s body, and are responsible for maintaining water balance in both off-host and on-host ticks (Sonenshine and Roe, 2013a;Sonenshine, 1991a). There are three types of acini (alveoli) in the female tick (Type I, II and III) (Fig. 1F) and 4 types in males (I-IV). Type I acini are agranular and attached directly to the anterior part (or proximal region) of the salivary duct. They have been associated primarily with off-host osmoregulation through the absorption of water after hygroscopic saliva is secreted during dehydration (Kim et al., 2017;Kim et al., 2019;Sonenshine, 1991a, b;Sonenshine and Roe, 2013a;Walker et al., 1985). Type II and type III acini are granular and more complex in structure, which shows drastic anatomical changes during feeding (Sonenshine and Roe, 2013a;Sonenshine, 1991a;Walker et al., 1985). Type II and type III acini have been associated primarily with water and ion excretion during feeding periods in addition to the production and secretion of bioactive molecules into their host (Kaufman and Phillips, 1973;Kim et al., 2019;Perner et al., 2018;Sonenshine and Roe, 2013a;Valenzuela et al., 2002).

It has been reported that tick salivary secretion is regulated through neuronal and hormonal signals (Sauer et al., 2000). The main regulator of the excretion and the influx of fluid into the lumens of the type II and III acini is dopamine which acts as a paracrine/autocrine hormone (Kim et al., 2014;Kim et al., 2019;Sauer et al., 2000). Two different dopamine receptors have been identified as the downstream physiological targets in type II and III acini: D1 receptor for the water-solute influx and InvD1L for the expulsion of water solute via pumping/gating (Šimo et al., 2011;Šimo et al., 2014). Experiments by heterologous receptor expression suggested that D1 is mainly coupled to cAMP, while InvD1L to Ca²⁺ elevation in the downstream signaling pathway (Sauer et al., 2000;Šimo et al., 2011;Šimo et al., 2014). As the upstream signal in the control of salivary glands, a number of neuropeptidergic projections reaching to the salivary glands have been described: SIFamide, Elevenin, and MIP (myoinhibitory peptide) (Šimo and Park, 2014). The locations of the neuropeptidergic varicosities and the receptors implicated that their modulatory functions for control of secretion and also for the feedback control of autocrine dopamine in the salivary glands. Overall, the salivary glands, playing a vital role in osmoregulatory processes in addition to contributing to vector competence as tick saliva is an important factor in pathogen transmission (Nuttall, 2019a, b;Valenzuela et al., 2002), are tightly controlled by neural and hormonal components for the activities.

Hindgut and Malpighian tubules

Malpighian tubules and hindgut are located at the terminal end of the alimentary canal with the major function as excretion of metabolic wastes (Sonenshine and Roe, 2013a;Sonenshine, 1991a). A pair of Malpighian tubules, long tubes with the dead ends, arises at the midgut-hindgut junction. The hindgut is in direct contact with the anus and wrapped by longitudinal and cross muscles. A number of neural projections innervate the hindgut, which are neurons immunoreactive to SIFamide and MIP. The neuropeptide SIFamide stimulates hindgut motility, which was antagonized by MIP in Ixodes scapularis, (Šimo and Park, 2014).

It has been reported that K⁺ is the major solute in the excretion being defecated through the hindgut, while Na⁺ is the major solute in the saliva during feeding periods (Kaufman and Phillips, 1973). In insects, the primary role of Malpighian tubules is the elimination of nitrogenous waste in the form of uric acid by the formation of hyperosmotic urine through the transport of K⁺ from the hemolymph into the tubular lumen that is energized by V-ATPase and the transport of Na⁺ out of the cell through Na/K-ATPase (Sonenshine and Roe, 2013a). Tick Malpighian tubules are also known to be the major site for the formation of nitrogenous waste in the form of guanine-rich excreta (Hamdy and Sidrak, 1982;Maddrell and O'Donnell, 1992;Sonenshine, 2013). The mechanism controlling the tick Malpighian tubules has not been yet studied, while insect Malpighian tubule has been intensively studied for the hormonal components and the mechanisms of V-ATPase mediated fluid transport in formation and secretion of the primary urine (Beyenbach, 2003;Maddrell and O'Donnell, 1992).

In other blood-feeding insects such as the kissing bug, potassium urate is formed in the Malpighian tubules, and further urine concentration is achieved in the rectal sac through the reabsorption of water before excretion (Pant, 1988;Sonenshine and Roe, 2013a;Wigglesworth, 1931). In ixodid ticks, hindgut excretion is primarily for the removal of potassiumrich excreta. Guanine has been suggested as the main nitrogenous component of excretion in addition to haematin and undigested blood. However, little is known about the molecular components regulating hindgut activity (Sonenshine and Roe, 2013a). Although imminent water losses occur through the cuticle, which accounts for a significant amount of water loss, natural water losses through Malpighian tubules and hindgut in the process of excretion of waste is also likely a significant as the target of osmoregulatory disruptor.

Dermal secretion through the cuticle

The phenomenon of dermal excretion through the cuticle in Ixodid ticks has long been observed, although the osmoregulatory implications of the physiology have not been investigated. Dermal secretion constitutes a significant amount of water, at least 4% of fluid from the body weight within seconds (Yoder et al., 2009), and it is known to occur through the numerous dermal glands located on the tick’s epidermal cell layer. Four different types of dermal glands and pore structures have been described in ticks, although their functions are yet controversial (Sonenshine and Roe, 2013a), and their morphology appears to vary in structure depending on the tick species. This characterization is based exclusively on the anatomy of the pore structures being non-setal associated with no innervation. The dermal glands are classified into four structural categories: sensilla auriformia, sensilla sagittiformia, sensilla hastiformia, and sensilla latcniformia, the latter being absent in adult ticks.

Sensilla auriformia in R. appendiculatus was thought to be a sensory organ proprioceptor (Walker et al., 2014), while other studies defined it as a dermal gland (Pavis et al., 1994;Yoder et al., 2009). Sensilla hastiformia, also known as type I gland or small gland (Fig. 2A), is found in alloscutum and scutum of both metastriate and prostriate ticks in all stages, but appears to be underdeveloped in the non-fed stage (Walker et al., 1996). While scnsilla sagittiformia, also known as type II gland or large wax gland (Fig. 2B), are exclusive to Metastriate ticks and have been found in the alloscutum, edge of scutum, and anal plates of fed Rhipicephalus (Yoder et al., 2009). Type II glands are known to produce secretions in ticks which are in engorging stage but also in questing (non-fed) ticks (Walker et al., 1996;Walker et al., 2014). The primary role of type I and type II glands is assumed to secret defensive compounds against predators and pathogens. These chemical components of the secretion include squalene and other unidentified compounds, which can be externalized through canal (pore) openings on the cuticle surface (Hackman and Filshie, 1982;Pavis et al., 1994), some of which are suggested to be acting as allomones against predatory ants. However, the functional studies of the components of dermal secretions has not been successful in showing the clear biological activities despite the predictions of the hypothetical functions (Yoder et al., 1993). The dermal secretion has been reported to be triggered through mechanical stimulation (Sonenshine and Roe, 2013a;Walker et al., 1996) and the mechanically induced secretion provided heat tolerance in R. sanguineus ticks (Yoder et al., 2009). Our recent study demonstrated the role of the dermal secretion in thermoregulation and osmoregulation. With the significant amount of water being secreted through this mechanism, it provides a promising target physiology for tick control.

Thermoregulation and tick dermal secretion

Higher animals rely on thermoregulation to maintain the homeostasis of metabolic functions (Crompton et al., 1978;Terrien et al., 2011). Thermoregulation in arthropods has been observed during their blood meal in order to prevent the heat stress caused by the warm ingested blood (Benoit et al., 2019). Different thermoregulatory strategies have been observed in several blood-feeding arthropods. Mosquitoes retain drops of urine in the abdomen which provides temperature decreases as the urine evaporates (Lahondère and Lazzari, 2012), while the kissing bug dissipates heat from the head during the blood meal (Lahondère et al., 2017). Arthropod thermoregulation has been broadly studied in hematophagous arthropods during the blood ingestion: however, studies on evaporative cooling in arthropods including ticks during the non-feeding periods is limited.

Dermal secretion of hard ticks has primarily been associated with defense mechanisms against pathogens and predators such as the predatory ant (Pavis et al., 1994;Yoder et al., 1993). Our research group has recently suggested that this secretion can also serve as a thermoregulatory strategy during the off-host periods. Heat stimulation induced dermal secretion of the unfed lone star tick A. americanum, followed by a rapid cooling of the body temperature. The secretion can be observed at temperatures as low as 35°C (similar temperatures during summer in the tick microhabitat) and at least 60% of consistently responding in different trials (Fig. 2C and 2D). Serotonin and Na⁺/ K⁺-ATPase have been proposed as the main regulators of dermal secretion. Injection of serotonin (1mM in 10nl) induced dermal secretion, which was inhibited by pre-injection with ouabain (a Na⁺/K⁺-ATPase blocker) (Fig. 2E). Previous reports show that the secretion of salivary glands and hindgut is controlled by the neural and hormonal controllers (Kim et al., 2019;Šimo and Park, 2014). Our recent findings on dermal secretion molecular controllers also suggest that the response is controlled by neural/hormonal mechanisms. The proposed thermoregulatory function of the dermal secretion is probably stemmed from the fact that the secretion accounts for an excessive loss of fluid (4.1% of body weight). In addition to the rapid response in secretion, which suggests it may be triggered by heat rather than just as a defense mechanism caused by disturbance. Thus, dermal secretory physiology likely occurs in the field when ticks are not able to avoid high temperatures, as suggested to occur in hot summer days periods .

Novel Strategy to Control Tick by Targeting the Vulnerable Osmoregulatory Physiology

In recent years, the research in ticks has significantly increased; however, the knowledge we currently have is yet limited in comparison to other blood-feeding arthropods such as mosquitoes. Control of ticks in the field is heavily relying upon the use of conventional insecticides and acaricides. Although the method of application and the formulation of toxic chemicals in tick control have been improved, new classes of chemicals for controlling ticks are urgently in need since ticks have developed acaricide resistance (Abbas et al., 2014;Coles and Dryden, 2014). The safety and environmental issues became more important in the development of tick control measures. Rational design of the tools and methods for tick control can be made based on understanding tick biology and physiology, and as such, targeting the vulnerable osmoregulatory physiology proposes a good target for management.

In metastriate ticks, water drinking behavior is common and has a significant impact on tick survival. Water drinking behavior is an important part of water conservation physiology and as such, it is a promising target for tick control. The delivery of toxic agents which leads to tick mortality, as in the case of phosphate-rich solution through drinking water, is a promising proposal for management and adds new avenues for tick research. In addition, the ingestion of bacteria, which can be later traced to the tick midgut, provides an opportunity for elucidating tick vector competence if certain bacteria can successfully colonize the tick gut.

Recent findings suggest that dermal secretion provides evaporative cooling, which could be occurring in nature. This could be a factor contributing to their extended survival rates and habitat expansions to geographical areas with hot temperatures. Dermal secretion involved in osmoregulatory physiology and thermoregulation may offer a vulnerable tick physiology that can be targeted for the development of a novel tick control strategy. Based on the prediction of the direct action of serotonin on the dermal gland, a candidate serotonin receptor (Aame5- HT1A) was recently identified from A. americanum (our unpublished data). Aame5-HT1A was found only in epidermalenriched carcass among the four different tissues including carcass, midgut, hindgut, and Malpighian tubules. The Aame5- HT1A orthologue was previously identified in R. microplus (the southern cattle tick) (Chen et al., 2004), but without its known function. The identification of potential agonistic or antagonistic compounds of these osmoregulatory and thermoregulatory controller molecules might provide an additional target for management.

Conclusion

Here we propose the use of new tick control strategies, which primarily target the vulnerable osmoregulatory physiology in Ixodidae ticks. The exploitation of tick water drinking behavior for the delivery of toxic agents (phosphates or bacteria), can induce tick mortality by dehydration or disruption of tick bacterial homeostasis.

The knowledge we have gained over the recent years about neuronal and hormonal control of the main tick osmoregulatory organs, combined with voluntary ingestion of toxic cagents, may improve our previous results by accelerating tick mortality. Similarly, through the delivery of osmoregulatory modulator molecules, blocking rehydration and/or inducing dehydration may accelerate tick death if combined with the delivery of other toxic agents. In this environmentally friendly manner, a tick-specific management strategy may be further developed and proposes a promising management strategy, alternative to pesticides, if applied in the field for tick control.