Iron metabolism in hard ticks (Acari: Ixodidae): The antidote to their toxic diet

Parasitology International 64 (2015) 182–189

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Parasitology International journal homepage: www.elsevier.com/locate/parint

Review

Iron metabolism in hard ticks (Acari: Ixodidae): The antidote to their toxic diet Remil Linggatong Galay a,b, Rika Umemiya-Shirafuji c, Masami Mochizuki a,b, Kozo Fujisaki d, Tetsuya Tanaka a,b,⁎ a

Department of Pathological and Preventive Veterinary Science, The United Graduate School of Veterinary Science, Yamaguchi University, Yoshida, Yamaguchi 753-8515, Japan Laboratory of Infectious Diseases, Joint Faculty of Veterinary Medicine, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan d National Agricultural and Food Research Organization, 3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856, Japan b c

a r t i c l e

i n f o

Article history: Received 27 October 2014 Received in revised form 1 December 2014 Accepted 9 December 2014 Available online 16 December 2014 Keywords: Ticks Iron Heme Iron-binding proteins Ferritin Hematophagy

a b s t r a c t Ticks are notorious parasitic arthropods, known for their completely host-blood-dependent lifestyle. Hard ticks (Acari: Ixodidae) feed on their hosts for several days and can ingest blood more than a hundred times their unfed weight. Their blood-feeding habit facilitates the transmission of various pathogens. It is remarkable how hard ticks cope with the toxic nature of their blood meal, which contains several molecules that can promote oxidative stress including iron. While it is required in several physiological processes, high amounts of iron can be dangerous because iron can also participate in the formation of free radicals that may cause cellular damage and death. Here we review the current knowledge on heme and inorganic iron metabolism in hard ticks and compare it with that in vertebrates and other arthropods. We briefly discuss the studies on heme transport, storage and detoxification, and the transport and storage of inorganic iron, with emphasis on the functions of tick ferritins. This review points out other aspects of tick iron metabolism that warrant further investigation, as compared to mammals and other arthropods. Further understanding of this physiological process may help in formulating new control strategies for tick infestation and the spread of tick-borne diseases. © 2014 Elsevier Ireland Ltd. All rights reserved.

Contents 1. 2. 3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A short review of blood digestion in hard ticks . . . . . . . . . . . . . . . . . . . . . Heme iron regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Heme transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Heme storage and detoxification . . . . . . . . . . . . . . . . . . . . . . . . 4. Non-heme iron regulation in hard ticks . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Host-transferrin movement in the hard ticks . . . . . . . . . . . . . . . . . . 4.2. Ferritins and their role in tick iron transport and storage . . . . . . . . . . . . . 4.3. Iron-regulatory protein and regulation of iron-binding proteins . . . . . . . . . . 4.5. Antioxidant and immune-related functions of iron-binding proteins in the hard ticks 5. Summary and future perspectives. . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction ⁎ Corresponding author at: Laboratory of Infectious Diseases, Joint Faculty of Veterinary Medicine, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan. Tel.: +81 99 285 3570. E-mail address: [email protected] (T. Tanaka).

http://dx.doi.org/10.1016/j.parint.2014.12.005 1383-5769/© 2014 Elsevier Ireland Ltd. All rights reserved.

Ticks are obligate blood-feeding ectoparasites of humans and animals, notorious for their significant role as vectors of several diseases worldwide. While the blood-feeding behavior of ticks, which causes

R.L. Galay et al. / Parasitology International 64 (2015) 182–189

anemia, irritation, allergic reactions, and (in the case of some tick species) toxicoses [1] can be already harmful in its own rite, particularly in heavy infestation, further harm is done because transmission of pathogens also occurs during this process [2]. Ticks are considered second to mosquitoes in terms of worldwide public health impact due to pathogen transmission [3]. They serve as vectors of several viruses, bacteria, and protozoa, which can usually infect multiple host species. Thus, understanding the different aspects of tick physiology, particularly those involved in pathogen acquisition and transmission, is critical in formulating an effective control strategy. Ticks have successfully adapted to an exclusively hematophagous lifestyle, relying completely on their host's blood as the sole source of nutrients needed for their survival and reproduction [4]. Similar to other hematophagous arthropods, they have developed several mechanisms that allow them to exsanguinate their hosts, evading hemostatic, inflammatory, and immune responses [5]. The saliva of ticks is an important arsenal for feeding, containing hundreds of pharmacologically active substances [6] that have anti-hemostatic [7], anti-inflammatory, and immunomodulatory functions [8]. This allows hard ticks to stay attached to their hosts for several days to complete their blood meal. Aside from their sophisticated feeding mechanism, the adaptation of ticks to potentially toxic molecules in their hosts' blood has enabled them to thrive in their exclusively hematophagous lifestyle. Remarkably, adult female hard ticks can ingest blood more than a hundred times their unfed body weight in a single blood meal [9]. Host blood contains pro-oxidants that may induce oxidative stress. A number of antioxidant enzymes have been identified in the tick midgut that likely are responsible for protecting ticks from oxidative stress [10,11]. Iron is another component of the host blood that has both beneficial and harmful effects on ticks. Whereas iron is an essential component of several proteins involved in fundamental biochemical activities including cellular respiration, energy metabolism, and DNA synthesis, it is also potentially toxic [12]. The breakdown of hemoglobin, the most abundant protein in mammalian blood that comes from red blood cells, produces heme iron that is known for its ability to generate reactive oxygen species (ROS) [13]. The non-heme, inorganic iron, ferric (Fe3+) transferrin, when liberated from host transferrin becomes ferrous iron (Fe2 +), which can also participate in the formation of ROS. Therefore, iron metabolism clearly must also have a crucial function that allows the ticks to survive and even thrive in their potentially toxic lifestyle. This review discusses current knowledge in the iron metabolism of hard ticks. We first briefly describe blood digestion and then the fates of heme and non-heme iron from the blood meal. We also discuss the functions of known iron-binding proteins in hard ticks and compare currently available information on the iron metabolism of vertebrates and other arthropods.

2. A short review of blood digestion in hard ticks Hard ticks have a longer feeding period compared to soft ticks (Acari: Argasidae) and other hematophagous arthropods, generally divided into a slow feeding phase after attachment, and a rapid phase shortly before engorgement [14]. Feeding may be completed in 7–10 days, depending on the tick species, developmental stage and length of starvation. For instance, females of the hard tick Haemaphysalis longicornis being maintained in our laboratory can complete a blood meal within 7 days on average [15]. Blood digestion has also been divided into three phases, consisting of two continuous digestion phases, the first after the onset of feeding and the second after detachment, and a reduced digestion phase during rapid engorgement. The digestion process is also unique in ticks because it occurs intracellularly within digestive cells [14]. Blood meal components are taken up by digestive cells of the midgut through absorptive receptor-mediated endocytosis [16] regulated by protein kinase C [17]. Host proteins, including hemoglobin, are broken down within the lysosomal vesicles of digestive cells. All types of

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digestive cells differentiate from a single cell type, the stem cell [18], exhibiting different stages of digestion [19]. Blood meal digestion involves a number of proteolytic enzymes [20], most of which target the hemoglobin molecule at specific sites [21]. In the hard tick Ixodes ricinus, the hemoglobinolytic activity in the midgut was found to increase from 4 days after attachment and peaked right after detachment from the host [22]. Hemoglobinolysis occurs at an acidic pH (3.5 to 4.5) initiated by cathepsin D, known also as longepsin in H. longicornis [23], with the aid of cathepsin L and legumain [21,24]. Other hemoglobinolytic enzymes, such as cathepsin B, including longipain of H. longicornis [25], serine proteases [26], serine carboxypeptidase [27], and leucine aminopeptidase [28], are involved in the further degradation of hemoglobin. During feeding, hematin from hemoglobin breakdown, together with other by-products of blood digestion, accumulates in large vacuoles of spent cells, which are excreted in the form of feces [18]. After detachment from the host, ticks defecate minimally, so heme is sequestered and aggregated instead into a specialized vesicle called hemosome [29]. 3. Heme iron regulation Heme is an important component of many proteins including cytochromes and enzymes that function in electron transport, cellular respiration, signal transduction and detoxification, but it can also promote the formation of free radicals in its free form [30]. In arthropods, including ticks, heme is incorporated into vitellin (Vn), a major yolk protein, and its precursor, vitellogenin (Vg) [31], both of which are critical in reproduction [32], the former serving as a nutrient reserve for the embryo and during starvation periods post-hatching [33]. In female mosquitoes, the majority of absorbed iron after the blood meal including heme was detected in the ovaries and eggs, implying that iron is an essential nutrient for reproduction and embryonic development [34]. In the arthropod vector of Chagas' disease, Rhodnius prolixus, heme binds to the Rhodnius heme-binding protein (RHBP ) in the hemolymph and is incorporated into the eggs, where it functions as a heme source for embryonic development [35]. While other hematophagus arthropods can synthesize heme on their own, the cattle tick Rhipicephalus (Boophilus) microplus was shown to have no means of synthesizing its own heme, and thus it relies solely on the heme recovered from its blood meal [36]. In this section, we review the transport and storage of heme in hard ticks in comparison to other hematophagous arthropods. 3.1. Heme transport After detachment from the host, the hemoglobin levels in the tick midgut lumen continuously drop until day 33 [16]. Using a fluorescent heme-analog Palladium-mesoporphyrin (Pd-mP) artificially fed to R. microplus adults, Lara et al. [37] followed the fate of hemoglobin in digestive cells. The uptake of hemoglobin by digestive cells was thought to occur through receptor-mediated endocytosis. After uptake, the fluorescence of Pd-mP was observed mainly in the large vesicles of digestive cells. Fluorescence in hemosomes was observed at 6 h after feeding. Hemoglobin digestion is concentrated in the luminal side, while heme aggregation is concentrated in the basal side of digestive cells. Heme can also cross the midgut barrier, although the mechanism is not yet clear, and bind to heme glycolipoproteins in the hemolymph [31] that will be further discussed in the next section. 3.2. Heme storage and detoxification Due to the potentially toxic effects of heme, hematophagous arthropods developed various mechanisms that protect them from excessive heme in their diet [38,39]. In mosquitoes, while a large amount of heme iron is excreted as waste, a substantial amount remains in the spent female at the end of their gonadotrophic cycle [34]. The

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peritrophic matrix in the midgut of the Aedes aegypti mosquito has been associated with heme detoxification due to its heme-binding function [40]. The triatomine bug R. prolixus also employs several mechanisms for heme detoxification. One is the aggregation of heme into hemozoin, previously recognized only in malaria parasites [41], which is induced by the perimicrovillar membranes in the insect's midgut [42] and has been also observed in other triatomine species [43]. Another is through the binding by RHBP in the hemolymph, mentioned above [44]. A heme degradation pathway involving heme oxygenase has also been demonstrated in both R. prolixus [45] and A. aegypti [46]. In hard ticks, the products of blood digestion accumulate in digestive cells [18] inside the organelles previously termed residual bodies [16]. In this early work, these residual bodies were found to have high concentrations of iron, most probably heme. Lara et al. [29] later confirmed that these residual bodies are accumulations of heme and termed them hemosomes, most likely serving as a heme detoxification mechanism. Under transmission electron microscopy, the hemosome structure showed a delimiting membrane with a compact core surrounded by a cortical region with several layers, which is distinct from hemozoin, being non-crystalline in nature. In female ticks, these hemosomes persist until the end of oviposition period, filling up most of the cytoplasm of digestive cells until they die. Heme-binding storage proteins in ticks, such as the hemelipoglycocarrier protein (CP), Vg, and Vn, also function in protecting the ticks from heme toxicity [31]. The CP characterized in Dermacentor variabilis [47] and D. marginatus [48] and the Heme Lipoprotein (HeLp) of R. microplus [49] are major storage proteins in the hemolymph that consist of two subunits binding heme transported from the midgut to the hemocoel. In D. variabilis, CP mRNA was strongly detected in the fat body and salivary glands of fed female ticks and weakly detected in the midgut and ovaries [50], whereas the protein expression of D. marginatus CP was detected in salivary glands but not in the midgut [48]. It remains unclear, however, whether CP is secreted in the saliva and if it has a role in tick feeding. An isolated HeLp molecule from R. microplus was found to contain two heme molecules, and a purified HeLp molecule can bind up to six additional heme molecules. Moreover, when radioactively labeled HeLp was injected into the hemocoel of female ticks and monitored over time, the radioactivity continuously decreased in the hemolymph from 30 min after injection but correspondingly increased in the ovaries, suggesting that HeLp might be incorporated into the ovaries. Interestingly, HeLp was not detected in the eggs after Western blot analysis [49]. Vg, the large multimeric precursor of the major yolk protein, Vn, in many oviparous animals, including arthropods [51], is synthesized by the fat body in ticks. Vg production is initiated by blood feeding and then transported to the oocytes in a process called vitellogenesis during rapid feeding [33]. Vn, which is responsible for the brown color of the eggs, is capable of binding up to 30 molecules of heme, functioning as a heme reservoir and antioxidant defense during embryonic development [52]. In H. longicornis, three cDNAs encoding for Vg (HlVg) were identified with specific organ distribution. Silencing of these HlVgs resulted in significant reductions in engorged body weight and egg production [32], confirming that Vg indeed plays a critical role in tick reproduction. Antioxidant enzymes have also been implicated in heme detoxification. Several antioxidant enzymes have been identified in the midgut of hard ticks, mostly expressed during the advanced stage of blood feeding [10,11]. Glutathione-S-transferase (GST) and its reduced form, GSH, have been particularly associated with heme catabolism [53], but this has not yet been demonstrated in hard ticks. Instead, inhibition of catalase in engorged R. microplus females by injection of 3-amino-1,2,4-triazole (AT) has been shown to inhibit heme aggregation, altering heme detoxification [54]. After injection of AT, digestive cells had high amounts of heme dispersed in the cytosol, in contrast to the control ticks wherein heme is confined in the hemosomes. Altered egg morphology, reduced egg laying, and high mortality were also observed in engorged ticks injected with AT. These suggest that the function of

catalase against hydrogen peroxide is related to heme detoxification in hard ticks, although the exact mechanism has not yet been clarified. 4. Non-heme iron regulation in hard ticks Non-heme iron accounts for the smaller proportion of circulating iron in the host blood compared to hemoglobin and is usually bound to metalloproteins, such as iron–sulfur clusters, or to an iron-binding protein transferrin as ferric-transferrin [55]. Compared to heme iron, ferric-transferrin is more bioavailable and a higher percentage of ingested ferric-transferrin is utilized by female mosquitoes, primarily stored in the eggs [34]. Several proteins have already been identified in mosquitoes [56,57] and Drosophila melanogaster [58] that function similarly to the proteins involved in mammalian iron metabolism, including the regulation of absorption, transport, and storage of nonheme iron obtained from their diet. In comparison, little is known about the regulation of non-heme iron in hard ticks. This section will discuss currently available information on the fate of ferric-transferrin from the host blood and the functions of iron-binding proteins, particularly ferritins, in hard ticks. 4.1. Host-transferrin movement in the hard ticks Transferrin (Tf) is a monomeric protein of around 76–81 kDa, with two structurally similar lobes that can bind a single Fe3+ atom each, functioning primarily in iron transport [55]. In mammals, Fe3 + from the diet is absorbed by the enterocytes through divalent metal transporter 1 (DMT1, also known as SLC11A2 and NRAMP2), and then exported into the blood circulation by the basolateral exporter ferroportin (SLC40A1). The multicopper oxidase hephaestin is responsible for the oxidation of Fe2+ that binds to plasma Tf. Diferric-Tf transports iron into different tissues, gaining entry into the cells through clathrin-mediated endocytosis following binding to Tf receptor 1 [59]. A Tf molecule, Tf1, has been identified in several species of insects, including hematophagous species, and is similar in structure to mammalian Tf. Differences have been noted in the Tf1 of hematophagous and non-hematophagous insects in terms of molecular weight and glycosylation. The Tf1 of hematophagous insects has a lower molecular weight and is non-glycosylated [60]. However, the role of insect Tf in iron transport and its uptake mechanism is still unclear [58,60]. In hard ticks, only partial sequences of putative Tf of I. scapularis (GenBank: EEC13740) and R. pulchellus (GenBank: JAA63555) have been identified, but their function in iron metabolism has not been investigated. Additionally, although a partial sequence for a putative transferrin receptor of I. ricinus (GenBank: JAB73833.1) has been identified, it remains to be elucidated whether it really serves a function similar to vertebrate transferrin receptors, and therefore warrants thorough investigation. Meanwhile, a number of studies showed that host Tf acquired from the blood meal can circulate within the ticks and can be maintained for long periods after their blood meal. The presence of host Tf was first demonstrated in the hard tick D. variabilis, detected in the hemolymph after blood feeding [61]. Host Tf was also among the host proteins detected in I. scapularis and Amblyomma americanum nymphs 3 months after molting [62]. Recently, it has been demonstrated in H. longicornis that host Tf can be transferred from the midgut to the oocytes via the hemolymph, persisting up to 28 days after the blood meal, although it was not detected in the eggs after oviposition [63]. The same study also showed that host Tf can be maintained by the ticks even after 3 months post-molting from one developmental stage to another. The movement of host Tf within the tick and its persistence for a long time demonstrated its importance as an iron source and suggested that it may have a role in iron transport within hard ticks, although it is unknown whether the host Tf retained its iron-binding ability. However, the exact mechanism of host Tf movement, particularly the uptake by oocytes, remains unclear. Since Tf uptake in

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mammalian cells is receptor mediated, it is possible that a yet unidentified receptor in the ticks can also facilitate the uptake of host Tf. 4.2. Ferritins and their role in tick iron transport and storage Ferritin (FER) is another iron-binding protein that is present in almost all organisms. A FER molecule typically consists of 24 subunits arranged in a bundle forming an almost spherical shell with a large cavity that can accommodate up to 4000 Fe3 + atoms [64]. Two types of subunits with a molecular weight of around 20 kDa each have been identified; the heavy (H) chain that comprises the ferroxidase center responsible for the oxidation of Fe2+ to Fe3+ and the light (L) chain responsible for the accumulation of Fe3 + in the FER core. The ratio of the H chain and L chain in vertebrate FER varies among tissues, depending on its role in iron metabolism. Homologues of these subunits have also been identified in insects, usually present in equal ratio, except in mosquitoes that have secretory FER consisting mainly of the H chain. Amino acid residues responsible for iron-binding and ferroxidase activity in the H chain are highly conserved among different species [65]. The primary function of FER is the storage of excess iron available in the cellular labile iron pool after delivery of Tf [55]. The iron storage process involves the binding and oxidation of Fe2 + in the catalytic centers; formation of a Fe3+ core in the cavity, which yields stable ferrihydrite nuclei; and further incorporation of Fe3+ into the nuclei [65]. Vertebrate FERs are generally classified as intracellular or secretory types. Intracellular or cytoplasmic FER is the predominant type, widely distributed in different tissues but particularly abundant in the liver and spleen. Secretory FER is present in a lesser amount compared to cytoplasmic FER, which is distributed mainly in body fluids, such as serum, cerebrospinal fluid and synovial fluids, and is useful as an indicator of body iron status and the presence of inflammatory condition and malignant diseases [66]. In contrast, FERs of insects are primarily secretory in nature, mainly associated within the vacuolar system, with the exception of Homopteran insects that have abundant cytoplasmic FER [67]. In contrast to its vertebrate counterpart, insect secretory FERs that are abundant in the hemolymph serve as iron transporters. In the yellow fever mosquito A. aegypti, dietary iron is loaded into FER that is transported to the eggs [34]. A third type, the mitochondrial FER, has been found in some organs in mammals and D. melanogaster [68], which was thought to play a role in supplying iron for the iron–sulfur enzymes and protection against oxidative damage [64]. In other invertebrates such as the snail Lymnaea stagnalis [69] and the schistosome Schistosoma mansoni [70], FERs are classified as either soma FER, which is an intracellular type found in most tissues except the oocytes, or yolk FER, which is a secretory type synthesized by the digestive gland and incorporated into the yolk in the oocytes. Hard ticks have both intracellular and a secretory FER, herein referred to as FER1 and FER2 respectively. FER1 was first characterized in I. ricinus, which is similar to the H chain of vertebrate FERs, with a molecular weight of 20–21 kDa per monomer, forming a 24-mer with a molecular weight of 510 kDa and having conserved motifs in the ferroxidase center [71]. Analysis of the full-length cDNAs of FER1 from eight species of hard ticks [72] revealed the presence of a conserved ironresponsive element (IRE) in the 5′ untranslated region found also in vertebrate FERs, which is involved in post-transcriptional regulation [73]. The sequence of this IRE was almost identical, and no signal peptide was found in any of the eight species examined. Multiple alignment of FER1 amino acid sequences of hard ticks showed at least 80% identity among different species. Western blot analysis showed that FER1 was consistently detected in all tick tissues examined but not in the hemolymph [71,74]. Whereas mosquito fer mRNA is up-regulated in response to blood meal or high levels of iron [75,76], tick fer1 mRNA levels do not change after blood feeding [71,74,77,78] or exposure to exogenous iron [79]. Protein expression analysis during blood feeding showed that the level of FER1 does not change in the midgut but increased in the salivary glands [74], suggesting that its post-transcriptional regulation is organ

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dependent. The constitutive expression of FER1 in the midgut implies its important role as the primary iron storage organ. The FER2 of hard ticks is also similar to the H chain of vertebrate FERs, each monomer with a 21 kDa molecular weight, but does not seem to form a stable 24-mer folding [74]. In contrast to the fer1, the fer2 sequence lacks an IRE, similar to the mRNAs of the L. stagnalis yolk FER [69], S. mansoni FERs [70], and Haliotis discus discus Abf1 [80]. FER2 is similar to insect secretory FER in that it contains a signal peptide and was detected in the hemolymph [74,77]. Whereas the fer1 level is constitutively expressed in all organs examined, the fer2 level in the midgut and hemocytes is unchanged during blood feeding but decreased in the salivary glands and fat body toward engorgement and was detected only in unfed and 1-day-fed ovary [74]. Similar to fer1, fer2 was not up-regulated by increasing iron levels [79], which is in contrast to the secretory FER of insects [76,81]. The presence of FER2 in engorged salivary glands, ovaries, and eggs despite the very low transcript level implied that it was transported through the hemolymph, probably from the midgut, since immunofluorescent localization and Western blot analysis demonstrated that FER2 is abundant in partially-fed midgut [74]. Furthermore, knockdown of fer2 resulted to the absence of FER1 in the salivary glands and ovary after blood feeding. These indicate that like the secretory FER of insects, FER2 functions as an iron transporter in hard ticks [77,79]. The crucial role of FERs in successful blood feeding and reproduction of hard ticks was demonstrated by gene silencing through RNA interference (RNAi) in two separate studies on I. ricinus [77] and H. longicornis [74]. After RNAi, female ticks failed to engorge and had reduced fecundity. High mortality was also observed in knockdowned ticks after detachment from their host without laying any egg. Morphological analyses of partially-fed midgut showed abnormalities in the digestive cells of knockdowned ticks, such as disrupted microvilli and cell membrane, vacuolated cytoplasm, and reduced hematin production [74]. Diminished hematin production indicates reduced digestive activity and also suggests that FERs are important in the normal function of digestive cells. In the insect D. melanogaster, knockdown of the midgut fer led to iron accumulation in the iron-cell region of the midgut but systemic deficiency, with retarded development and lowered survival rate [82]. Knockdown of fer genes also seemed to have an indirect effect on the expression of Vg genes that affected the reproductive capacity of the ticks. Silencing of fer1 in H. longicornis resulted in the silencing of HlVg-1 and reduction of HlVg-3, while silencing of fer2 resulted in reduced expression of HlVg-1 and HlVg-3 [74]. This effect on HlVgs, and tick reproduction in general, is most likely due to the failure of fer-silenced ticks to attain the critical weight required for vitellogenesis [83] and the inadequate nutrients, especially amino acids that activate vitellogenesis [84]. Similar to the secreted FER of mosquitoes [34] and the yolk FER of snails [69] and schistosomes [70], FER2 appears to play an important role in embryonic development in hard ticks, serving as a source of iron or protecting the embryo from iron toxicity. FER2 was expressed throughout the embryogenesis and fer2-knockdowned ticks laid eggs with abnormal morphology that failed to hatch, strongly indicating that FER2 is required for normal embryonic development [74]. 4.3. Iron-regulatory protein and regulation of iron-binding proteins The iron-regulatory proteins (IRPs) are cytoplasmic RNA-binding proteins that respond to changes in cellular iron availability by binding to the IRE in the mRNA of Tf and fer, regulating the translation of these iron-binding proteins [85]. When the level of iron in the cells is low, the IRP binds to the IRE in the 5′ terminal of fer, preventing its translation, and to the IRE in the 3′ terminal of Tf blocking its degradation. Conversely, when there is a surplus of iron in the cells, the IRP does not bind to the IRE, promoting the translation of fer and degradation of Tf [59]. Two IRPs have been identified in mammals, IRP1 and IRP2, the former having a second function as a cytoplasmic aconitase [85]. A single homologue of IRP1 has been identified in the mosquito Anopheles gambiae

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Host Tf Fe3+

Hb (RBC)

Fe3+

DMT1?

Dcytb?

Tfr?

Tfr?

endocytosis

Host Tf Host Tf

Hb (RBC)

MCO? DMT1?

Fe2+

Fe2+

Fe2+

Fe2+

Fe2+

breakdown

Fpn?

Hm

FER1

Hm

Hemosome

Tick Tf?

Labile Fe pool

HO?

Fe3+

Hm

Fe3+

Fe3+

storage

FER2

export

Hm

accumulation

MIDGUT (DIGESTIVE CELL)

H E M O L Y M P H

Fe3+

Hm

FER2

Vn storage

CP

OVARY (OOCYTES)

Fig. 1. Proposed model for iron metabolism in hard ticks. Red blood cells (RBCs) are taken up by digestive cells and digested intracellularly, releasing hemoglobin (Hb). Hb breakdown produces heme (Hm), which accumulates in the hemosome or passes through the midgut for export, binding to carrier proteins (CP). The carrier proteins may be transported to peripheral organs, including the ovary, where Hm may be incorporated into vitellin (Vn). Meanwhile, non-heme iron from host transferrin (Tf) undergoes a different pathway. After the uptake of ferric iron (Fe3+) loaded host Tf, iron is released within the digestive cell as ferrous iron (Fe2+) that accumulates in the labile Fe pool. These Fe2+ atoms can be stored within the intracellular Ferritin1 (FER1) or loaded into Ferritin2 (FER2), which can be secreted into the hemolymph and transported to other organs such as ovary. Conversely, host Tf may also pass through the midgut and circulate within the hemolymph and may reach the other organs. Unknown aspects of tick iron metabolism are also presented here. First, the uptake mechanism of host Tf by digestive cells and other cells, and its movement to other organs are still unclear. Second, presence of a functional tick Tf and transferrin receptor (Tfr) and their roles in iron transport are still unknown. Third, the presence and role of other iron-binding proteins similar to those in mammalian iron metabolism (enclosed in dashed boxes), such as divalent metal transporter 1 (DMT1), duodenal cytochrome b (Dcytb), hephaestin or multicopper oxidase (MCO), ferroportin (Fpn) and heme oxygenase (HO), require further investigation. Dashed arrows denote unknown pathways.

[57], while two IRP1-like proteins have been identified in D. melanogaster [58], although only 1 of these homologues, IRP1-A, has IRE-binding activity [86]. In the hard tick I. ricinus, a single homologue of IRP1 has been characterized [77]. Silencing irp1 increased the translation of fer1 and lowered the hatching of eggs, although no significant effects on blood feeding and oviposition were observed.

4.5. Antioxidant and immune-related functions of iron-binding proteins in the hard ticks Due to their iron-binding function, keeping Fe2+ from reacting with hydroxyl radicals that may produce reactive oxygen species (ROS), thus preventing oxidative stress, Tf and FER are classified as secondary

Table 1 Proteins involved in human iron metabolism that have not yet been studied in hard ticks. Human protein

Function

Duodenal cytochrome b

Reduction of Fe3+ for intestinal uptake

NRAMP2/DMT1 Transferrin receptor Hephaestin (multicopper oxidase) Ferroportin Heme oxygenase a

2+

Transport of Fe across the apical membrane of enterocytes Facilitation of transferrin entry into the cell Re-oxidation of Fe2+ to Fe3+ for export outside the cell Export of iron Heme degradation

Candidate tick homologuea

GenBank accession number

Identity (%)a

Putative cytochrome B561 (Ixodes scapularis) Putative transport system membrane protein (I. scapularis) Putative transferrin receptor, partial (I. ricinus) Hypothetical protein IscW_ISCW012230 (I. scapularis) (None) Ca2+ sensor (I. scapularis)

EEC07620.1

41

EEC01792

63

JAB73833.1 XP_002413022

27 40

EEC01522.1

38

Using Basic Local Alignment Search Tool (BLAST) analysis of the National Center for Biotechnology Information (NCBI) online software, the candidate tick homologue was selected based on identity (http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome).

R.L. Galay et al. / Parasitology International 64 (2015) 182–189

antioxidants [87]. The response of FERs to oxidative stress is well known in mammals [88,89] and mosquitoes [90]. In hard ticks, this function of FER was demonstrated in H. longicornis [79]. The oxidative stress status of fer-knockdowned ticks was evaluated after a blood meal or exposure to high levels of iron by determining the levels of malondialdehyde produced during lipid peroxidation and carbonyl protein produced during protein oxidation. High levels of malondialdehyde and carbonyl protein were seen in the whole bodies of fer-knockdowned ticks after a blood meal and iron injection as well as in the engorged midguts of ferknockdowned ticks, indicating that iron overload in the absence of FERs promoted ROS production and, consequently, oxidative stress. The antioxidant function of insect Tf has been demonstrated in the larvae of the beetle Protaetia brevitarsis, wherein Tf mRNA and protein expression was up-regulated following exposure to hydrogen peroxide, and increased hydrogen peroxide production after Tf RNAi [91]. The function of iron-binding proteins in immunity is based on the principle that most pathogens require iron for their growth and compete with their hosts in utilizing this essential metal [92]. To limit the access of pathogens to iron, the hosts developed iron-sequestration mechanisms, primarily through iron-binding proteins, which have been well established particularly in vertebrates [93–95]. FER of insects was implicated in response to bacterial challenge as demonstrated in the mosquito A. aegypti [96], the parasitoid wasp Asobara tabida [97] and the bumblebee Bombus ignitus [81]. Insect Tf was also associated with immune response, not only against bacteria [98,99], but also to fungi [91] and other pathogens [100,101]. In hard ticks, knowledge of the role of iron-binding proteins in immunity is scant. In a study on D. variabilis FER, its mRNA was up-regulated following an Escherichia coli challenge [78]. In our laboratory, studies on H. longicornis FERs show that the silencing of fer genes decreases the survival of ticks when challenged with E. coli and allows bacterial multiplication in the hemocytes [Galay et al., unpublished data]. Further studies are needed to elucidate this function of iron-binding proteins in hard ticks. 5. Summary and future perspectives Currently, we have a modest understanding of tick iron metabolism compared to what is known about other arthropods [57,58]. This review presented the few studies on hard ticks related to iron metabolism, most of which dealt with heme utilization and detoxification. The critical importance of iron metabolism in the physiology of hard ticks has already been demonstrated by the silencing of fer genes in two hard tick species [74,77]. After combining the available knowledge on these studies, we propose a model for tick iron metabolism (Fig. 1). Heme and non-heme iron undergo separate pathways. Aggregation of large amounts of heme within the midgut is a unique detoxification mechanism in ticks. Meanwhile, non-heme iron is acted upon by FERs. In this model, we also identified other aspects of iron metabolism that remain to be elucidated. For instance, the functions of tick Tf, particularly its involvement in iron transport, still require investigation. Our previous study on H. longicornis showed that iron injected in the hemocoel stimulated FER expression in the midgut, including that of the digestive cells in the luminal side, suggesting that iron traffic in the midgut may be two-way (i.e., from luminal side to basal side, which happens after blood digestion, and from basal side to luminal side) [79]. Furthermore, the mechanism for the movement of host Tf in the ticks is still puzzling. In mammals, iron traffic in the cells is facilitated by a transferrin receptor and ferroportin, neither of which has yet been studied in the ticks. In our model, we also included homologues of proteins involved in vertebrate iron metabolism or counterpart tick proteins with similar functions (Table 1) that are yet to be identified and investigated. These include duodenal cytochrome b (Dcytb) and divalent metal transporter 1 (DMT1) involved in uptake of dietary iron by mammalian enterocytes [12], multicopper oxidase, which facilitates the oxidation of Fe2 + to Fe3+ and is essential in iron export from the digestive organ, not only in mammals but also in D. melanogaster [102,103], and heme oxygenase

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that catalyze heme oxidation in the presence oxygen and NADPH [39]. Furthermore, the iron metabolism of soft ticks is also poorly understood. The more exciting aspect of research on tick iron metabolism lies in its implication for tick and pathogen control. Due to the dependence of ticks on host blood for survival, targeting the mechanisms that allow them to evade toxicity that can result from the high iron content of their diet seems to be an attractive control strategy. Studies on the use of recombinant FER2 against tick infestations showed promising results [104,105], and its efficacy may be increased when used in combination with another antigen as an anti-tick vaccine. Furthermore, the relationship between tick iron metabolism and pathogen infection is an interesting topic, and understanding it may help in blocking pathogen transmission.

Acknowledgments This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Numbers 25292173 and 26660229, a cooperative research grant (26-joint-6) from the National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, and the Morinaga Foundation. RL Galay is supported by the Japanese Government Ministry of Education, Culture, Sports, Science, and Technology Scholarship (Monbukagakusho: MEXT) for doctoral fellowship.

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