circulatory system and hemolymph

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CIRCULATORY SYSTEM AND HEMOLYMPH Structure, Physiology, and Molecular Biology LIBOR GRUBHOFFER, N ATALIIA RUDENKO, MARIE VANCOVA, MARYN A GOLOVCHENKO, AND JAN STERBA

1. introduction Tick hemolymph is the primary means of nutrient and hormone transport, maintains osmotic pressure, and fights injuries and pathogens with the help of hemocytes while allowing the dissemination of successfully surviving pathogens. One would anticipate a tremendous amount of available information about this system; however, there is still a lot of information missing. This chapter tries to compile the most important knowledge on the morphology of the circulatory system and its components, as well as on their composition and function.

2. morphology and ultrastructure of the tick circulatory system The morphology of the tick circulatory system (CS) of Argasidae and Ixodidae was described in detail by Obenchain and Oliver (1976), and a comprehensive summary was given by Sonenshine (1991). The CS is composed of the heart suspended by muscles in the dorsal anterior region of the body, the aorta, periganglionic and anterior sinuses, and arterial vessels (Figs. 11.1–11.6). Arteries

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Circulatory System and Hemolymph 259

FIGURE 11.1: Schematic diagram of the circulatory system in ixodid ticks. From the pericardial sinus,

hemolymph enters the pulsatile part of the heart through paired ostia. The heart pumps the hemolymph into the aorta and periganglionic sinus. The hemolymph is then directed by arterial sinuses and arteries into other parts of tick body. A, aorta; Aomc, aortic myocardial cone; As, anterior sinus; Dlsm, dorsolateral suspensory muscles; Hpp, pulsatile part of the heart; Lso, lateral segmental organ; O, ostia; Pa 1–4, pedal arteries; Pcsp, pericardial septum; Pgs, periganglionic sinus; Syn, synganglion; Vlsm, ventrolateral suspensory muscles. From Binnington, K.C. and Obenchain, F.D. (1982) Structure and function of the circulatory, nervous, and neuroendocrine systems of ticks. In F.D. Obenchain and R. Galun (Eds.), Physiology of Ticks. Oxford, UK: Pergamon Press, 277–350, with permission from the author.

facilitate the directional transport of the hemolymph into different hemocoele spaces. The CS is tightly connected with the synganglion, the central nervous system of the tick. The morphology of the CSs of Argasidae and Ixodidae differs in several details, such as the shape of the heart in diastole, the extent of tracheation, and the presence of an endosternum in Argasidae. The dorsal anterior sinus is better developed in Ixodidae than in Argasidae (Obenchain and Oliver 1976).


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260 BIOLOGY OF TICKS

FIGURE 11.2: Scanning electron micrograph illustrating the morphology of the circulatory system of the

Ixodes ricinus male tick. The circulatory system is located in the middle of the dorsal side of the tick body. The main components of the circulatory system are the heart (H), the aorta (A), periganglionic (Pgs) and arterial sinuses, and pedal vessels (Pa). The aorta directs hemolymph from the heart into the periganglionic sinus that encloses the synganglion (Syn). Extensions of the sinus form pedal arteries. Sg, salivary glands; Md, midgut diverticulum. Measurement bar: 100 μm.

2.1. structure (and function) of the heart The principal organ of the CS, the heart, is localized in the space between the dorsal cuticle and the midgut region (Fig. 11.2). It is anteriorly bordered by cheliceral retractor muscles and is posteriorly surrounded by secretory foveal gland tissue in metastriate ticks (Obenchain and Oliver 1976). The heart is fastened into the dorsolateral (dlsm) and ventrolateral (vlsm) suspensory muscle apparatus (Figs. 11.4, 11.6) and is surrounded by an extensive system of tracheae and tracheolae. The vlsm forms long branches that are connected to the ventral side of the heart (Fig. 11.6). These muscles were found to be associated with nerve endings that provide motor innervation. The dlsm is arranged in groups and probably determines the shape of the heart during the diastole phase of the cardiac cycle, when the heart shape is pentagonal (Ixodidae) (Fig. 11.4) or subtriangular (Argasidae). The dlsm and vlsm are highly branched in the proximity of the pericardial septum (Fig. 11.4) and are associated with the connective tissue of the septum, which contains numerous perforations through which the hemolymph flows. The septum creates a connection between the heart and the dorsal part of the tick cavity. Connective tissues of the septum function as a support membrane for pericardial cells (Figs. 11.4–11.5) (Obenchain and Oliver 1976). The heart is a dorsoventrally flattened sac-like structure that is composed of several layers. Its outer sheet, the pericardial septum, consists of a layer of mesenchymal cells interwoven densely with trachea (Fig. 11.7) (Obenchain and Oliver 1976). The layer is a continuation of the layer of the surrounding the synganglion (periganglionic sinus). The myocardium, an important inner layer of the heart, is composed of striated muscle. Based


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Ixodes ricinus male tick. The median notch (white arrow) on the dorsal surface of the midgut diverticula (Md) accommodates the aorta (missing here). The black arrow points out the entrance of the aorta to the periganglionic sinus. Measurement bar: 10 μm.

on the orientation of the muscle fibers, the heart is divided into 2 regions that differ in function: (i) the aortic myocardial cone is formed from the longitudinal striated fibers in the anterior part of the heart, and (ii) the circularly oriented muscles form a pulsatile part in the posterior region of the heart (Fig. 11.7) (Binnington and Obenchain 1982). In the posterior part of the heart, the muscles are perforated ventrolaterally by 2 pairs of openings, the ostia (according to Balashov [1972], there is only 1 pair in the case of Argas persicus). Ostia allow the hemolymph to flow from the pericardial sinus to the inside of the heart. The ostia are formed with muscular lips whose contraction during the systolic phase leads to closing of the ostia and movement of the hemolymph forward to the adjacent cardiac compartments (Obenchain and Oliver 1976).


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FIGURE 11.4: Scanning electron micrograph of the dorsal myocardium (M), the pericardial septum

(Pcsp), and associated dorsolateral suspensory muscles (Dlsm) in an Ixodes ricinus male tick. Measurement bar: 10 μm.

FIGURE 11.5: Scanning electron micrograph showing a pericardial cell attached to the pericardial

septum in an Ixodes ricinus male tick (enlarged view of Fig. 11.4). Measurement bar: 1 μm.

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Ixodes ricinus male tick. Ventral view of the heart; branches of ventrolateral suspensory muscles (Vlsm) attach to the pericardial septum (Pcsp). Pc, pericardial cells. Measurement bar: 10 μm.


Ixodes ricinus male tick. Ventral view of the inside of a damaged pericardial sinus (Pcsi). M, myocardium; Pcsp, pericardial septum. Measurement bar: 10 μm.

2.2. structure and function of arterial vessels and sinuses The main arterial vessel, the aorta, arises from the anterior part of the heart and opens into the periganglionic sinus surrounding the synganglion. This arrangement enables sufficient perfusion of the synganglion by hemolymph. The aorta is located below the cheliceral retractor muscles in a median notch of the dorsal surface of the midgut (Fig. 11.3). The aortic valve lies between the aortic-myocardial cone and the aorta. The inner layer of the aorta is formed by longitudinal muscle fibers. The organization of the muscle fibers differs from that in the pulsatile part of the heart. The aorta is covered with an


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264 BIOLOGY OF TICKS outer sheet composed of 1 layer of membrane-bound mesenchymal cells. Similar structures were shown to form walls of pericardial, periganglionic, and anterior sinuses and pedal arteries. In contrast to the aorta, the sinus wall does not contain muscle fibers (Obenchain and Oliver 1976). A ventral extension of the periganglionic sinus surrounds the pedal nerve trunks and thus encloses the spaces that function as pedal arteries. Four pedal arteries at each side of the tick body direct hemolymph to the legs (Figs. 11.1, 11.2). Suspensory extrinsic muscles are associated with the periganglionic sinus and pedal arteries and influence the arterial pressure. The periganglionic sinus continues anteriorly and forms the ventral and dorsal anterior sinuses. Palpal and stomodeal nerves are present in the center of the ventral (periesophageal) anterior sinus. The dorsal anterior sinus surrounds the cheliceral and optic nerves and leads directly to the mouthpart region. Increased hemolymph pressure in the dorsal anterior sinus allows the movement of chelicerae and the hypostome during feeding (Obenchain and Oliver 1976).

3. hemolymph composition and function Tick hemolymph is a complex fluid composed of plasma (usually clear to brown in color) and hemocytes (circulating cells). Hemolymph surrounds all internal tissues and organs and serves as an exchange medium for the transport of nutrient molecules, hormones, and products of cellular metabolism. Local changes in hemolymph pressure are important in the ventilation of the tracheal system, in thermoregulation, and in molting (Gullan and Cranston 2010). It is involved in protection from physical injury (e.g., the wound-healing process that involves hemocytes and plasma coagulation) and pathogen invasion (the immune response). The amount of hemolymph in arthropods varies among species and depends on the developmental and feeding stage. A total hemolymph volume calculated for the land crab Cardisoma guanhumi was assumed to be 30% of its body weight (Burggren et al. 1990). The volume of hemolymph in soft-bodied mosquito larvae ranges from 20% to 40% of their body weight (Gullan and Cranston 2010). In ixodid ticks, the hemolymph volume accounts for about 20% to 30% of the body weight (Kaufman and Phillips 1973). Compared to vertebrate blood, tick hemolymph contains relatively high concentrations of proteins, lipids, carbohydrates, amino acids, pigments, organic acids, and inorganic ions, which make the major contribution to the total hemolymph osmolality (Araman 1979). Hemolymph composition can be altered after the consumption of a blood meal or through the influence of temperature changes or the presence of pathogens (Mullins 1985). Recently, mRNA-based and proteomics approaches have been employed to reveal differentially expressed proteins in the hemolymph of challenged ticks, allowing the investigation of tick innate immunity at the protein level (Stopforth et al. 2010).

3.1. plasma proteins in ticks Plasma composes approximately 90% of tick hemolymph, and proteins constitute its major soluble component (11.5%–14.3% by weight) (Gudderra et al. 2002). In spite of the importance of ticks as disease vectors, our knowledge about tick hemolymph proteins is limited relative to similar knowledge about other arthropods, particularly insects (Gudderra et al. 2002). For example,


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a two-dimensional map of 160 hemolymph proteins of Drosophila melanogaster has been constructed for investigation of the changes that occur at the protein level in different developmental stages, in different physiological conditions, or after infection (Vierstraete et al. 2003). In addition to enzymes and hormones, hemolymph proteins associated with tick immunity represent a variety of proteins secreted into the plasma in response to physical injury, blood feeding, or invasion of the tick body by pathogenic or nonpathogenic microorganisms. They are termed “humoral factors” and represent a wide variety of antimicrobial peptides (AMPs), lysozymes, defensins, histidine-rich peptides, coagulation factors, lectins, non-cationic peptides, alphamacroglobulin-like glycoproteins, proteases and their inhibitors, and other recognition molecules (reviewed in Taylor 2006; Sonenshine and Hynes 2008).

3.1.1. Vitellogenins and other heme-lipoproteins Vitellogenin, the precursor of vitellin, represents one of the major proteins in females at the time of egg development (11% of the total hemolymph protein) (Thompson et al. 2007). Vitellogenin is a hemelipoglycocarrier protein synthesized by the fat body (FB), gut cells, and, to a lesser extent, ovaria of tick females (in some species) after mating and feeding. It is secreted into the hemolymph and then is transported and incorporated into eggs as vitellin. The heme in vitellogenins is derived from the digestion of host hemoglobin (Gudderra et al. 2002). Vitellogenins have been studied and partially characterized in Ornithodoros moubata (Chinzei 1983; Chinzei and Yano 1985), O. parkeri (Taylor et al. 1991), Hyalomma dromedarii (Schriefer 1991), Dermacentor variabilis (Thompson et al. 2007; Khalil et al. 2011), Ixodes scapularis (James and Oliver 1997), Haemaphysalis longicornis (Boldbaatar et al. 2010), and Rhipicephalus microplus (Granjeno-Colin et al. 2008). Other major heme-binding proteins are the carrier proteins (CPs) and hemelipoglycoproteins (HLGPs). In contrast to vitellogenin, CPs are found in each developmental stage of female and male ticks. These proteins were detected and named DvCP or HLGP in D. variabilis (Guddera et al. 2001; Donohue et al. 2008), HeLp in R. microplus (RmCP) (Maya-Monteiro et al. 2004), and HLGP in D. marginatus (Dupejova et al. 2011). CPs are high-molecular-weight dimeric proteins (approximately 340 to 400 kDa) that contain 3 motifs found in tick vitellogenins: (i) a lipoprotein N-terminal domain, (ii) a domain of unknown function, and (iii) a C-terminal von Willebrand factor type D domain (Donohue et al. 2008). Further information on vitellogenins, vitellin, and CPs can be found in Chapter 15.

3.1.2. Defensins and other antimicrobial peptides The key element of innate immunity is the speed with which defense responses occur. One of the facets of tick defense is the rapid and transient synthesis of a battery of potent AMPs following infection or trauma. AMPs and other immune-related proteins are secreted proteins synthesized within the FBs or hemocytes. The activity spectrum of the immune peptides is diverse. The most studied and representative group of tick AMPs are the defensins, cationic peptides with 6 cysteines that have been identified in the hemolymph of many insects and other invertebrates. The defensins form the first line of host defense against pathogens. They are classified into 3 major groups: (i) peptides with an α-helical conformation (cecropins, magainins, etc.), (ii) cyclic and open-cyclic peptides with pairs of cysteine residues (defensins, protegrins, etc.), and


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266 BIOLOGY OF TICKS (iii) peptides with an overrepresentation of some amino acids (proline rich, histidine rich, etc.). Despite extreme diversity in their primary and secondary structures, all natural AMPs are active against gram-positive or gram-negative bacteria, fungi, yeast, viruses, and protozoa. Arthropod defensins were first isolated from cultured cells of the flesh fly, Sarcophaga peregrine, by Matsuyama and Natori (1988). All invertebrate defensins have the same cysteine pairings, Cys1-Cys4, Cys2-Cys5, and Cys3-Cys6, that stabilize the molecule and maintain the tertiary structure, known as the “defensin fold” (Ganz 2003). Defensins are small peptides approximately 4 kDa in size. Together with signal peptide and prepro regions, which are separated by an “RVRR” sequence from the mature region, defensins are about 67 to 92 amino acids long. Mature peptides usually range from 36 to 46 amino acids in length (Bulet et al. 2004) and are highly conserved. To date, tick defensins have been identified in nearly 20 hard and soft tick species (Rudenko et al. 2005, 2007; Sonenshine and Hynes 2008; Saito et al. 2009; Chrudimska et al. 2010). Varisin, the major hemolymph defensin in D. variabilis, was initially identified in tick hemocytes (Johns et al. 2001). Being active against gram-positive bacteria, varisin is active against B. burgdorferi. In combination with chicken lysozymes, it killed 65% of the borrelia spirochetes in 1 hour, indicating a possible synergism between varisin and lysozymes (Johns et al. 2001; Sonenshine et al. 2002). The role of varisin in the innate immunity of D. variabilis was confirmed by RNAi. The activity of varisin knock-down tick hemolymph against the gram-positive bacterium Microccocus luteus was reduced by 50%. This fact suggests that varisin is important, but not vital, for the antimicrobial activity of D. variabilis hemolymph (Hynes et al. 2008). An unexpected effect of varisin silencing was the reduced infection of D. variabilis with the rickettsial pathogen Anaplasma marginale (Kocan et al. 2008). A second defensin, defensin 2, was recently isolated from D. variabilis midgut. It appears to be phylogenetically distinct from most of other tick defensins. Its role in innate immunity is unclear (Ceraul et al. 2007). Two defensin isoforms, def1 and def2, were identified from another hard tick, I. ricinus (Rudenko et al. 2005, 2007; Chrudimska et al. 2010). Both def1 and def2 inhibit the growth of gram-positive bacteria (M. luteus, B. subtilis, S. aureus) in very low concentrations (minimum inhibitory concentration [MIC] of 0.37–50 μM) but show no activity against borrelia. The defensin isoforms differ by only 1 amino acid in their mature peptide sequence, but the antimicrobial activity of the def2 isoform (found in each tick tissue) is at least twice as strong as that of def1 (midgut specific) (Chrudimska et al. 2010). Alignment of the mature region showed similarities to defensins from other species of hard ticks ranging from 77% for I. scapularis to 56% for A. hebraeum. The similarity to the 4 already described defensins (omdef-A to omdef-D) from the soft tick O. moubata was 61% to 63% in the mature peptide. Analysis of the genomic structure of I. ricinus defensin genes showed the presence of 3 exons and 2 introns; this was the first evidence of an intron/exon defensin structure in the hard tick (Rudenko et al. 2007). The partial sequence of a third I. ricinus defensin, def 3 (AAT46066), was described as well. It contains 4 of the 6 essential Cys residues and is clearly different from def1 and def2 at both nucleotide and amino acid levels. The nucleotide and deduced amino acid sequences of this partial defensin show greater similarity to defensins from D. variabilis and H. longicornis than to those from I. ricinus (Rego 2005). A sequence matching the tick defensins was found in the salivary glands of I. scapularis (Valenzuela et al. 2002). Later, a novel defensin, scapularisin, was identified in the midgut, hemocytes, and FBs of I. scapularis (Hynes et al. 2005). Its mature peptide showed 78.9% similarity to the mature hemolymph defensin from D. variabilis. The role of scapularisin as an AMP is unclear, as


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it does not seem to contribute to the tick immune defense. B. burgdorferi incubated with I. scapularis hemolymph remained viable and active. The finding that I. scapularis has a defensin that does not control B. burgdorferi suggests that other unknown factors are important in the antimicrobial defense in this tick (Hynes et al. 2005). A sequence similar to scapularisin was found in the whole body cDNA library of I. persulcatus. Its expression was induced by blood feeding in all tick developmental stages. It is unknown whether I. persulcatus defensin is secreted into hemolymph. Commercially synthesized putative mature I. persulcatus defensin markedly inhibited the growth of gram-positive bacteria, inclu ding Staphylococcus aureus (MIC = 0.8 μM), Bacillus subtilis (MIC = 0.5 μM), and Corynebacterium rendle (MIC = 0.3 μM), but not of gram-negative bacteria, with the exception of Escherichia coli O157 (Saito et al. 2009). Genes encoding 2 non-cationic defensin-like peptides were isolated from a differentially expressed cDNA library of A. hebraeum synganglia. In addition, an Amblyomma defensin peptide-2 was purified from the hemolymph of blood-fed tick females and showed antimicrobial activity against E. coli and S. aureus, with MICs of 30 and 7.5 μM, respectively. No antifungal activity was found (Lai et al. 2004a). Amblyomma defensin peptide-2 has a predicted net negative charge, a unique feature among all other known defensin-like peptides. The mechanism of interaction between Amblyomma defensin 2 and bacteria is unclear (Lai et al. 2004a). One of the possibilities is the employment of an alternative mechanism, such as the stimulation of autolytic enzymes, the inhibition of DNA synthesis, and interference with bacterial DNA and/or protein synthesis (Wu et al. 1999). Recently, a defensin (americin) transcript was found in the hemocytes, midgut, FBs, and salivary glands (SGs) of A. americanum (Todd et al. 2007). The mature americin peptide showed 44.7% and 42.1% similarity to anionic Amblyomma defensin peptide-1 and peptide-2, respectively. But deeper analysis of sequential similarities and tissue expression patterns revealed that americin is much more closely related to the D. variabilis (varisin) and I. scapularis (scapularisin) defensins than to defensins of A. hebraeum. Similar tissue distributions of defensins in non-competent and competent vectors indicate that anti-Borrelia activity in non-competent vectors may be triggered by either post-transcriptional regulation of defensin or the activity of a different set of proteins (Ceraul 2005). Defensin also is expressed in the hemocytes and FBs of R. microplus (Fogaca et al. 2004). Containing 6 conserved cysteines, a 4.29 kDa peptide shows high similarity to the insect defensin family members. The tissue expression pattern of R. microplus defensin is closest to that of the Ornithodoros moubata defensin D isoform (Nakajima et al. 2003b). Defensins have been identified in the soft tick O. moubata, in which 4 different isoforms (A, B, C, and D) have been characterized (Nakajima et al. 2001, 2002). The 4 isoforms appear to be divided into 2 types: 1 type shows primary expression in the midgut (A, B, and C), and the other is primarily expressed in the FBs (D). Gene expression of defensin A, B, or C in the FBs is weak relative to expression in the midgut. In contrast, defensin D gene expression in the FBs is stronger than in the midgut. It seems that the 2 types of Ornithodoros defensins are involved at different ratios in the immunity of the hemolymph and the midgut (Nakajima et al. 2003b). Analysis of the active functions of synthetic Ornithodoros defensin showed the presence of bactericidal and antibacterial activity against many gram-positive bacteria, but not against gram-negative bacteria, and low hemolytic activity characteristic of invertebrate defensins (Nakajima et al. 2003a). In addition to classical defensins, a large number of other peptides with antimicrobial activity have been identified in arthropods. Microplusin, a 10.2 Da polypeptide with 6 conservative cysteine residues, was isolated from the hemolymph of R. microplus (Fogaca et al. 2004). It belongs to a new type of AMP family with


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268 BIOLOGY OF TICKS multiple histidines and a novel secondary structure composed of 4 to 6 α-helices. The recent functional analysis showed that microplusin chelates copper ions by binding them, likely to histidines. Microplusin was active against gram-positive bacteria, filamentous fungi like Aspergillus niger, and yeast C. neoformans, but not C. albicans. The detected bacteriostatic effect of microplusin against Microccocus luteus was probably caused by its ability to bind copper ions, which are vital for bacterial respiration (Silva et al. 2009). Almost simultaneously, another histidine-rich AMP, hebraein, was isolated from the hemolymph of fed females of A. hebraeum. This 11 kDa anionic protein has 6 cysteine residues, an all-α-helical structure, and a histidine-rich carboxyl-terminal region. Hebraein has the same cysteine motif as microplusin, and they are 62% identical and 73% similar. Both proteins possess the same antimicrobial pattern. Antifungal activity was greatly reduced in a histidine-deficient mutant, indicating that the histidine residues are important for antifungal activity (Lai et al. 2004b). Further information on AMPs can be found in Chapter 5 of Volume 2.

3.1.3. Lysozymes The fact that some other antimicrobial activities are present in tick hemolymph was confirmed by multiple studies. RNAi studies of D. variabilis defensin revealed that after its inactivation, tick hemolymph still possessed significant activity against M. luteus. The most probable candidate in tick hemolymph capable of inhibiting the growth of M. luteus was presumed to be lysozyme(s) expressed by hemocytes (Simser et al. 2004a). The synergetic role of lysozymes in enhancing varisin antimicrobial activity supported this assumption (Johns et al. 2001). Lysozymes are ubiquitously expressed enzymes that lyse bacteria by cleaving the β-1,4 glycosidic bonds between N-acetyl muramic acid and N-acetyl glucosamine in the peptidoglycan layer of the bacterial cell walls. Ticks utilize host proteins as a defensive tool against invasive pathogens. For example, rabbit lysozyme has been purified from the hemolymph of Ornithodoros ticks (Hetru et al. 1997). Two hemolymph factors of D. variabilis showed activity against B. subtilis (Johns et al. 1998). One of these factors, a 14.5 kDa peptide, was identified as a lysozyme. Reports characterizing lysozymes in ticks have been based on circumstantial evidence such as the lysozyme-like immunoreactivity of hemocytes of I. ricinus (Kuhn and Haug 1994). The outcomes of these reports revealed that purified lysates from various tick species display lysozymelike activities in response to bacterial challenges (Johns et al. 1998). The lysozyme lytic activity of hemolymph, gut contents, and homogenates from the soft ticks Alveonasus lahorensis and O. papillipes was tested against that of M. lysodeikticus. Lysozyme activity increased 8- to 10-fold only in ticks challenged with different bacteria (Podboronov and Berdyev 1991; for an English review, see Podboronov 1991). Recently, a c-type lysozyme from H. longicornis (HlLysozyme) was characterized (Tanaka et al. 2010). HlLysozyme was detected in all tick developmental stages. Gene expression of HlLysozyme was up-regulated in the FBs, midgut, ovaries, and hemolymph of females after bacterial challenge, suggesting a possible role in the tick immune response to bacterial invasion. The facts presented support the theory that tick lysozyme is an enzyme with both immune and digestive characteristics that plays an important role in the innate immune responses of both soft and hard ticks (Kopacek et al. 1999; Grunclova et al. 2003). Comparative analyses of the highly conserved lytic peptides from D. variabilis, O. moubata, and H. longicornis identified them all as c-type lysozymes that share conserved common structural features, namely, 8 cysteine residues that form 4 disulfide bridges and conserved catalytic sites comprising glutamic acid and asparatic acid residues (Tanaka et al. 2010).


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3.1.4. Lectins Lectins are proteins capable of recognizing carbohydrates and are known to function as patternrecognition molecules in invertebrate immune mechanisms or as mediators of biological activity in cells (Vasta et al. 1994). Most lectins that have been isolated from arthropods are from the hemolymph (Mullins 1985; Vasta et al. 1999). The history of lectin studies in ticks is relatively brief. The earliest studies were done on hemolymph lectins of O. tartakovskyi, O. tholozani (papillipes), A. polonicus, and I. ricinus (Grubhoffer et al. 1991). Partially characterized lectins revealed their affinity to sialic acid and N-acetyl-D- glucosamine. Later, the binding activity of a novel 85 kDa lectin from I. ricinus with sialic acid, D-galactose, and N-acetyl-D-glucosamine was revealed (Kuhn et al. 1996). These findings led to the suggestion that lectins were the molecular factors of self- and non-self-recognition and defense in ticks, mediating, for example, the coiling phagocytosis of B. burgdorferi (Kuhn et al. 1994). No other lectin studies in ticks were recorded until the isolation of Dorin M, a sialic-acidbinding lectin from the hemolymph of the tick O. moubata (Grubhoffer and Kovar 1998) that is possibly involved in the tick immune system (Kovar et al. 2000). Dorin M shows sequence similarity to lectins from the horseshoe crab Tachypleus tridentatus, Tachylectins 5A and 5B (Kawabata and Tsuda 2002). Tachylectins are the major hemolymph lectins in the horseshoe crab and belong to a family of fibrinogen-related domain-containing proteins involved in immunity within vertebrates and invertebrates (Wang et al. 2004). The use of a bioinformatics approach coupled with molecular techniques resulted in the identification of a clearly different fibrinogen-related protein, OMFREP, in O. moubata hemocytes that was 65% identical to Dorin M. Two newly identified related molecules, Ixoderin A and Ixoderin B, were identified in I. ricinus. Both showed significant homology to Dorin M. Ixoderin A shares a similar expression profile. Ixoderin B was shown to be constitutively expressed only in the SGs of I. ricinus, with very marginal expression in the hemocytes (Rego et al. 2005). Studies revealed high similarity at the protein level of OMFREP and Ixoderin A to vertebrate immune proteins known as ficolins. Ficolins consist of a collagen-like domain and a C-terminal fibrinogen-related domain and structurally resemble mannose-binding lectin (Matsushita and Fujita 2001) that is known to have opsonic activity in serum, as well as the capacity to activate complement. Immune sera directed against Dorin M and other tick lectins have been used for the identification of novel lectins in other tick species (Sterba et al. 2011). A novel galectin (OmGalec), a protein with galactose-binding properties, was isolated from O. moubata. OmGalec has no significant similarity to OMFREP or Dorin M. It consists of tandem repeated carbohydrate recognition domains, in which the typical motifs important for carbohydrate affinity are conserved (Huang et al. 2007). This might lead to the modulation of hemocyte aggregation during infection or cross-linking with other cell types (Pace et al. 2002).

3.1.5. Macroblogulins The expression pattern of Dorin M is very similar to that of another plasma protein, an α2macroglobulin from O. moubata (TAM) (Kopacek et al. 2000). It is produced by hemocytes and is significantly expressed in the SGs after a blood meal. It was shown that TAM accounts for about 3% of the total plasma proteins of O. moubata. The defense role of TAM via the inhibition of microbial proteases (virulence factors of invading pathogens) is known to be an efficient mechanism in innate immunity (Armstrong 2001). Because of its high expression in the SGs, the role of TAM as a potential anti-coagulant should be considered.


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270 BIOLOGY OF TICKS Primary structure and functional analysis of α2-macroglobulin from I. ricinus (IrAM) showed that it is closely related to TAM (Buresova et al. 2009). IrAM is expressed in all tick developmental stages and tissues (except the midgut), and the protein is mainly present in the hemolymph. RNAi silencing of IrAM significantly reduces phagocytosis of the gram-negative bacteria Chryseobacterium indologenes by tick hemocytes. This bacterium is highly pathogenic to the soft tick O. moubata, but it is harmless to I. ricinus (Buresova et al. 2006). IrAM silencing had no effect on the phagocytosis of B. burgdorferi or gram-positive Staphylococcus xylosus. Such strict specificity in the reduction of phagocytosis might be mediated by the interaction of IrAM with C. indologenes metalloprotease, the major virulence factor for this pathogen (Pan et al. 2000).

3.1.6. Proteases and protease inhibitors The involvement of proteases in tick immunity is not clear, and knowledge of their role in tick hemolymph is limited. The arthropod clip-domain family serine proteinases consist of both an N-terminal disulfide knotted regulatory domain and a trypsinogen-like catalytic domain in the C-terminus (Jiang and Kanost 2000). A clip-domain serine proteinase homologue was identified in D. variabilis (Simser et al. 2004b). It was most similar to T. tridentatus hemocyte antimicrobial factor D and highly homologous to a number of immune-responsive gene products of arthropods, including insect prophenoloxidase-activating co-factors. Analysis of the expression pattern revealed that hemocytes were the primary site of its expression, and the level of expression was slightly higher in fed adults than in unfed. The orthologue of the D. variabilis clip- domain molecule was identified in D. andersoni embryonic cell line DAE100. It was shown that both tick proteases are present at steady-state levels within granules of tick hemocytes as “stored” immunity effectors and are released upon pathogen invasion (Simser et al. 2004b). Tick hemolymph contains proteins with serine protease inhibitory activity (serpins), and these inhibitors may exist in plasma or in hemocyte granules (Kanost and Jiang 1996). The serine protease inhibitors in tick hemolymph are likely involved in protection from infection. Some of them might inhibit fungal or bacterial proteases, whereas others probably contribute to the regulation of endogenous proteases involved in coagulation, prophenol oxidase activation, or cy tokine activation (Polanowski and Wilusz 1996). Serpins might represent some of the most interesting antigens for the development of an anti-tick vaccine because of their potential functions as inhibitors in arthropod homeostasis (Mulenga et al. 2000). A novel cysteine-rich peptide (Ixodidin) was isolated from the hemocytes of R. microplus. In addition to antimicrobial effects, this peptide exhibited proteolytic inhibitory activity against 2 exogenous serine proteinases, chymotrypsin and elastase. It is most likely that Ixodidin exerts different actions in tick hemolymph, acting directly against invasive pathogens, mediating other immune responses in the R. microplus hemocoel, and preserving its own integrity and the integrity of other AMPs (Fogaca et al. 2006).

3.1.7. Oxidoreductases Hemocytes of R. microplus have been shown to produce reactive oxygen species (ROS) upon phagocytosis (Pereira et al. 2001). Two separate thioredoxin peroxidase (peroxiredoxin) genes were strongly induced in the hemolymph, SGs, and midgut of I. ricinus after B. burgdorferi infection (Rudenko et al. 2005). It is possible that the I. ricinus peroxiredoxin gene is induced in


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response to oxidative stress as a consequence of B. burgdorferi infection. This could be a result of ROS released by tick hemocytes as part of the antibacterial response (Pereira et al. 2001). In addition, ticks encounter ROS generated by vertebrate immune cells such as neutrophils during blood feeding (Tsuji et al. 2001). Another gene identified in I. ricinus after a B. burgdorferi– infected blood meal with a possible role as an antioxidant is glutathione S-transferase (GST) (Rudenko et al. 2005). GST belongs to a gene family involved in the detoxification of xenobiotic compounds, including insecticides. It was observed that GST genes are often induced by ROS. This represents an adaptive response, as these enzymes detoxify some of the toxic metabolites produced within the cell by oxidative stress (Hayes and Strange 2000).

3.1.8. MD-2-related lipid-recognition domain-related proteins Expression of the gene encoding Der-p2 allergen-like protein in I. ricinus is induced by a blood meal (Horackova et al. 2010a). I. ricinus Der-p2 belongs to a diverse family of MD-2-related lipid-recognition (ML) proteins that includes major allergens of house dust mites, human MD2, or similar proteins from D. melanogaster. Similar genes or their protein products have not yet been characterized in any other tick species. Analysis of the expression pattern showed that Derp2 is strongly induced in the hemolymph and gut. The ability of Der-p2 to interact with IgE might indicate its involvement in the tick immune response. The predicted three-dimensional structure of I. ricinus Der-p2 showed the presence of a cavity between the plates formed by the β-sheets for lipid binding. This feature was confirmed in several members of the ML protein family. Although the characterization of Der-p2 is not yet complete, the protein most probably participates in the recognition and transport of lipids from blood or in the recognition of surface patterns created by lipidic molecules of microorganisms (Horackova et al. 2010a). A second gene encoding ML-domain-containing protein (IrML) was strongly induced in I. ricinus by a blood meal (Horackova et al. 2010b). Tick guts were defined as a primary site of IrML expression, but it was detected in hemolymph and SGs in all tick developmental stages. Analysis of the predicted structure of I. ricinus ML-domain-containing protein and its localization in the tick body suggests that IrML is a secreted protein and is possibly involved in tick innate immunity (Horackova et al. 2010b).

3.2. other hemolymph compounds regulating osmolality and ph

In addition to the wide range of proteins with diverse functions discussed above, other compounds make up tick hemolymph (minerals, lipids, hormones, etc.). The volume of hemolymph changes corresponding to each of the tick life stages, as well as during feeding. However, the proportion of the hemolymph in the tick body does not change significantly in the course of feeding. In A. arboreus, the hemolymph volume increases from 3.8 μl (28.5% of the whole body) in unfed females to 8.57 μl (25%) on engorgement day and then decreases to 6.81 μl (22.5%) on oviposition day. In A. persicus, these values are slightly higher, and the hemolymph represents 31%–38% of the whole body (Hefnawy 1972a). In D. andersonii, the hemolymph volume constituted about 23% of the total body weight in unfed and feeding ticks (Kaufman and Phillips 1973).


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272 BIOLOGY OF TICKS The content of the most abundant ions was studied in D. andersonii. Overall, hemolymph osmotic pressure and ion concentration decreased during tick feeding. In unfed ticks, the ion concentration was approximately 280 mM Na+, 20 mM K+, and 170 mM Cl−. The sodium concentration decreased on day 3 of tick feeding to 160 mM Na+ and remained stable throughout the remainder of the feeding process. On feeding day 5, ion concentrations declined to 7.5 mM for K+ and 125 mM for Cl−. The osmotic pressure decreased from approximately 527 mOsm/l during the first day of feeding to about 375 mOsm/l on the fifth day of feeding (Kaufman and Phillips 1973). Hemolymph osmotic pressure changes have been studied in detail in A. persicus and A. arboreus. In both cases, the unfed tick hemolymph osmolality was 390 mOsm/l. On the day of feeding, but before coxal fluid emission, the concentration decreased to 365 and 342 mOsm/l in the 2 species, respectively. Following coxal fluid emission, the osmolality declined to 310 and 322 mOsm/l, respectively. The osmotic pressure rose again after the completion of feeding; 1 day after feeding, the values were 370 and 360 mOsm/l, respectively, and 1 day after oviposition, the pressure exceeded the values prior to feeding and rose to 454 and 491 mOsm/l for A. persicus and A. arboreus, respectively (Hefnawy 1972b). The fatty acid content of hemolymph was studied in H. dromedarii and A. excavatum ticks. In both ticks, the concentration patterns of fatty acids were similar. A total of 20 fatty acids were identified with carbon chain lengths ranging from C2 to C24. Palmitic, stearic, oleic, and linoleic acids were present in pre-molting nymphs and engorging and ovipositing females and con stituted 80% to 90% of the total fatty acids (Hajjar 1972). As for free amino acids, gamma- aminobutyric acid, glycine, serine, glutamine, alanine, glutamate, aspartate, and taurine were detected in tick hemolymph (Lucien et al. 1995). Free sugars were detected in tick hemolymph, predominantly glucose, and the presence of mannose, inositol, trehalose, and sorbitol was confirmed (Levenbook et al. 1980; Binnington and Obenchain 1982; Hwang 2006). Glycerol is part of tick hemolymph, playing a protective role against injuries induced by low or high temperatures. The normal concentration of glycerol in D. variabilis is 19 mM. Similarly, sorbitol seems to play a protective role in ticks: its concentration under normal conditions is negligible, but it increases to 1.3 mM during cold hardening (Hwang 2006). Low levels of antioxidants were found in tick hemolymph; however, there has been minimal research in this area (Graça-Souza et al. 2006). Antioxidants probably serve as protectants against the oxidative damage caused by an excess of heme from a blood meal. Protoheme was found in the hemolymph of fed D. andersonii females at a concentration of 0.46 μg/μl (Hamdy et al. 1974). The information regarding the source of heme is confusing. Whereas heme synthetic activity was reported in D. andersonii (Hamdy et al. 1974), the same pathway was shown to be inactive in B. microplus, and this tick relies solely on host heme supplies (Braz et al. 1999). Hemoglobin fragments with possible antimicrobial activity are present in tick hemolymph (Fogaca et al. 1999). Freely soluble hemocyanin was found in the hemolymph of the tick A. cajennense (Carneiro and Daemon 2002).

3.3. hemolymph clotting and immune defense Hemolymph clotting is a defense mechanism in arthropods that occurs in response to injury or pathogen invasion. Hemolymph clotting was described in D. variabilis as a process accompanied by granulocyte degranulation (Eggenberger et al. 1990). Several potential clotting factors


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have been described (Mulenga et al. 2001, 2003). The presence of platelet-derived growth factor and transforming growth factor, which are implicated in wound healing (Matsuo et al. 2007), was confirmed in hemocytes and other tick tissues.

4. tick hemocytes Hemocytes are freely circulating cells in the hemolymph. Attached hemocytes were found in association with arterial vessels, FBs, nephrocytes (Kuhn and Haug 1994), and SGs (Sterba et al. 2011).

4.1. classification Mature hemocytes are divided into several types based on their morphology and function (phagocytic ability). The classification system for hemocytes is the subject of considerable controversy. Three basic morphological types (i.e., prohemocytes, plasmatocytes, and granulocytes) are present in hard and soft ticks (Figs. 11.8–11.12). So far, only putative proliferation sites of hemocytes or hemocyte reservoirs have been described in wounded or infected ticks. Mitotic and

FIGURE 11.8: Ultrastructure of the circulating hemocytes of the ticks Ixodes ricinus and Ornithodoros

moubata. Granulocyte type I with electron-dense granules (dg) and granules with a tubular structure (sg). Black arrows point out invagination pits; secondary lysosomes are identified by white arrows. Measurement bar: 1 μm. From Borovickova, B. and Hypsa, V. (2005) Ontogeny of tick hemocytes: a comparative analysis of Ixodes ricinus and Ornithodoros moubata. Exp. Appl. Acarol. 35:317–333, with permission from Experimental and Applied Acarology.


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moubata. Granulocyte type II. Measurement bar: 1 μm.


moubata. Plasmatocytes. Measurement bar: 1 μm. From Borovickova, B. and Hypsa, V. (2005) Ontogeny of tick hemocytes: a comparative analysis of Ixodes ricinus and Ornithodoros moubata. Exp. Appl. Acarol. 35:317–333, with permission from Experimental and Applied Acarology.

proliferative activities of mature hemocytes (as observed during the engorgement of I. ricinus females) indicate that hemocytes can divide and be differentiated directly from the free circulating population of hemocytes (Kuhn 1996; Borovickova and Hypsa 2005). Granulocytes and plasmatocytes are 2 abundant, freely circulating hemocyte types in hard ticks. Two morphologically different types of granulocytes (granulocyte types I and II) have


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moubata. Granulocyte type II. dg, electron-dense granules. Measurement bar: 1 μm. From Borovickova, B. and Hypsa, V. (2005) Ontogeny of tick hemocytes: a comparative analysis of Ixodes ricinus and Ornithodoros moubata. Exp. Appl. Acarol. 35:317–333, with permission from Experimental and Applied Acarology.

been distinguished, predominantly on the base of granule morphology (Eggenberger et al. 1990; Kuhn and Haug 1994; Borovickova and Hypsa 2005; Habeeb and El-Hag 2008), in I. ricinus, I. scaluparis, D. variabilis, H. dromedarii, and H. asiaticum. However, in D. andersoni, Brinton and Burgdorfer (1971) distinguished 4 types of spherule cells (former name for granulocytes) and, moreover, disputable oenocytes (oenocytoids). In soft ticks, only 1 type of granulocyte, with morphological similarity to granulocyte type II of hard ticks, was described in O. moubata (Inoue et al. 2001; Borovickova and Hypsa 2005), O. erraticus (El Shoura 1989), and Argas arboreus (El Shoura 1986). Aside from the 3 basic types of hemocytes, unique granular hemocytes were observed in O. moubata and were classified as spherulocytes based on their morphology, which is similar to the lepidopteran hemocytic type (Cook et al. 1985; Borovickova and Hypsa 2005). However, this granulocyte type was not observed in previous studies that involved immature tick stages of Ornithodoros and Argas genera (El Shoura 1986, 1989).

4.2. structure The important morphological features that help to distinguish the type of hemocytes are the quantity, size, shape, presence of filopodia in, and nucleus/cytoplasm ratio of cells and the size, shape, quantity, and substructure of inclusion bodies. However, differences exist between the ultrastructures of immature and mature stages of hemocytes.


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moubata. Plasmatocyte. m, mitochondria; dg, electron-dense granules; hb, heterogeneous bodies. Lysosomes are identified by white arrows, and multivesicular bodies by the black arrow. Measurement bar: 1 μm. From Borovickova, B. and Hypsa, V. (2005) Ontogeny of tick hemocytes: a comparative analysis of Ixodes ricinus and Ornithodoros moubata. Exp. Appl. Acarol. 35:317–333, with permission from Experimental and Applied Acarology.

Prohemocyte-like cells were found occasionally in the hemolymph of engorged I. ricinus females and more often in the hemolymph of both larval and nymphal ixodid ticks (Balashov 1972). Attached prohemocytes were found to be associated with connective tissues (Kuhn and Haug 1994). The cells are small and round to oval (7 μm × 6 μm) with a large nucleus, numerous mitochondria, and several small granules (Borovickova and Hypsa 2005). Type I granulocytes in I. ricinus are large round or highly pleomorphic cells (6 μm in length) that contain variable electron-dense granules with homogeneous or tubular substructures (Fig. 11.8). A typical feature of the type I granulocytes is the presence of lysosomes and filopodia (Kuhn and Haug 1994). Type II granulocytes of hard ticks (12 μm × 9 μm) contain numerous electron-dense granules about 2 μm in diameter (Fig. 11.9). Different electron densities of central and peripheral areas were observed in condensing immature granules (Borovickova and Hypsa 2005). Type II granulocytes of soft ticks (Fig. 11.11) resemble morphologically the type II granulocytes of Ixodidae (Borovickova and Hypsa 2005). Plasmatocytes are the predominant hemocytes in ixodid ticks (Fig. 11.10) and vary in size (11 μm × 9 μm, along the long axis up to 30 μm) and shape. Granular inclusions are absent; the presence of primary lysosomes and secondary lysosomes accommodating engulfed material, lipid droplets, and numerous filopodia is typical. Plasmatocytes of O. moubata (Fig. 11.12) and A. arboreaus (El Shoura 1986) are mostly rounded or ovoid cells (10 μm × 12.5 μm) that contain a few small electron-dense granules and vacuoles. Filopodia are present infrequently.


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moubata. Spherulocyte with characteristic large granules in different stages of development (stars). Measurement bar: 1 μm. From Borovickova, B. and Hypsa, V. (2005) Ontogeny of tick hemocytes: a comparative analysis of Ixodes ricinus and Ornithodoros moubata. Exp. Appl. Acarol. 35:317–333, with permission from Experimental and Applied Acarology.

A large proportion of the hemocytes in O. moubata represent spherulocytes (11 μm × 14 μm) (Fig. 11.13). These cells are characterized by numerous electron-lucent granules with a tubular substructure that fill almost the entire cell content (Borovickova and Hypsa 2005). Oenocytes were observed in limited numbers only in D. andersoni hemolymph. The cells have an ovoid shape (18.3 μm × 11.9 μm) with numerous pronounced infoldings of the plasma membrane and contain granules measuring 0.4 μm in diameter (Brinton and Burgdorfer 1971).

5. molecular basis of hemocyte function The immune system provides ticks with quite an efficient defense against invasive pathogens. The defense responses of ticks to microorganisms are mediated via the immune effector cells (hemocytes) and guided by regulatory mechanisms of gene expression. The hemocyte populations in the hemolymph are dynamic and directly influenced by pathogen invasion. Pathogen invasion induces an increase in hemocyte number and their migration toward the pathogen. The typical hemocyte count is approximately 103 to 104 cells/μl (Johns et al. 1998; Ceraul et al. 2002), but the number increases rapidly in response to bacterial challenge, supporting the role of hemocytes in the phagocytosis of bacteria and yeasts (Loosova et al. 2001). For example, challenge of D. variabilis with spore-forming B. subtilis led to a 6.4-fold increase in hemocytes within 48 hours (Johns et al. 1998). The response of D. variabilis to infection with B. burgdorferi was even faster, with the amount of hemocytes increasing 3.1 times during the first


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278 BIOLOGY OF TICKS hour after the challenge. Lysis and removal of spirochetes from the system resulted in a rapid decrease of the hemocyte population; cell numbers dropped to normal levels within 24 hours (Johns et al. 2000). The cellular response to infection includes phagocytosis, encapsulation, and nodulation. Phagocytosis by hemocytes is a primary cellular defense response to microbial infection in several ticks (Kuhn and Haug 1994; Zhioua et al. 1996). In hard ticks, phagocytic activity has been reported mainly for plasmatocytes and type I granulocytes, and rarely for type II granulocytes. It seems that the diverse types of hemocytes that occur in the adult tick differ in their functional contribution to the tick immune system (Kuhn and Haug 1994; Borovickova and Hypsa 2005). The process of phagocytosis and the fate of the engulfed material are affected by many factors such as opsonization (opsonizing factors may originate from the tick or the host serum) or the ability to recognize pathogens. Inoue et al. (2001) proved that the phagocytosis of polystyrene beads by tick hemocytes was enhanced by blood-meal components such as complement. All these factors and the production of AMPs (Section 3.1.1) consequently influence the vector competence of tick species to transmit pathogens (Johns et al. 2001; Sonenshine et al. 2005). Hemocytes of the immunotolerant Ixodes ticks showed a slow phagocytic response to B. burgdroferi, in contrast to hemocytes of D. variabilis, which possess bactericidal factors that influence the phagocytic process and lead to the killing of bacteria (Johns et al. 2000, 2001). Tick (Ixodes spp.) hemocytes use 2 different mechanisms in the phagocytosis of B. burgdorferi: conventional and coiling phagocytosis (Fig. 11.14) (Rittig et al. 1996; personal observation). In some cases, cellular defense is diminished by the invading pathogen; for example, the intracellular

FIGURE 11.14: Transmission electron micrograph illustrating a coiling type of phagocytosis of Borrelia

burgdorferi B31 spirochetes by isolated tick hemocytes of Ixodes ricinus. Arrow shows the formation of pseudopods around engulfed bacteria (asterisk). Measurement bar: 200 nm. Photo courtesy of K. Zacharovová.


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parasite Rickettsia ricketsii is engulfed but not destroyed by plasmatocytes, which then serve as a route of dissemination from the gut to other tick tissues (Socolovschi et al. 2009). Lysosomal compartments are well developed, especially in plasmatocytes, and to a lesser degree in granulocytes of both types (Kuhn and Haug 1994; Inoue et al. 2001). Expression of the c-type lysozyme in hemocytes of D. variabilis was shown to be upregulated by gram-negative bacteria (E. coli) but was not influenced by blood meal (Simser et al. 2004a). Similarly, blood meal did not increase the expression of lysozymes in O. moubata hemocytes (Grunclova et al. 2003). Up-regulation of the c type lysozyme in hemolymph was observed in H. longicornis after infection (Tanaka et al. 2010). Tick hemocytes produce a number of AMPs such as defensins and cystatins, which help the tick to fight infection (Section 3.1.2). In addition to lysozyme and protease, granulocytes and plasmatocytes of R. microplus produce ROS after phagocytosis (Pereira et al. 2001), which leads to the destruction of pathogens. Protein–carbohydrate interactions are believed to be involved in pathogen recognition. Lectin molecules expressed on the surface of hemocytes can be utilized by ticks for this purpose (Grubhoffer et al. 1997, 2005). Several lectins expressed by hemocytes and other cells have been identified in ticks to date (Grubhoffer and Kovar 1998; Kovar et al. 2000; Rego et al. 2005; Huang et al. 2007). The ability of hemocytes to recognize lipopolysaccharides on a pathogen surface brings up another mechanism of microbial control in ticks: nodulation. Upon exposure to bacteria, plasmatocytes cluster, forming a bacteria-hemocyte microaggregate that prevents hemolymph circulation and results in adhesion to different organs within the hemocoel (the nodulation process). Nodulation is similar to encapsulation (see below), but without melanization, and probably without the layering of flattened dead and dying plasmatocytes. Nodule formation is the quantitatively predominant cellular defense reaction to bacterial challenges, responsible for clearing the largest proportion of pathogens (up to 90%) from circulation (Ceraul et al. 2002). Encapsulation is another mechanism used by arthropods to clear the hemocoel, particularly for gram-negative bacteria or for organisms too large to be engulfed via phagocytosis (Eggenberger et al. 1990; Ceraul et al. 2002). Encapsulation is a biphasic event that starts with the degranulation of type I granulocytes and hemolymph coagulation and continues with the formation of the capsule. The capsule is formed by both types of granulocytes and flattened plasmatocytes (Eggenberger et al. 1990). In insects, hemocytes surround abiotic objects with consecutive layers of dead and degranulating cells, with melanization as the final step (Gillespie et al. 1997).

6. future perspectives Considering the importance of hemolymph for tick survival, hemolymph’s involvement in vital processes in all life stages and in blood feeding, and the role of hemocytes in pathogen survival and transmission, the CS of ticks should attract far more interest from researchers in the future. There is a significant gap between the information currently available about the morphology of the CS and that about the function of its components. The rapidly expanding employment of mass spectrometry, transcriptomics, and genomics should provide a more thorough description of the tick proteome and other hemolymph components in different life stages and in response to the environment, hosts, and infection. The identification of


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280 BIOLOGY OF TICKS molecules present only in ticks and not in their vertebrate hosts (e.g., ferritin-2) (Hajdusek et al. 2010) might provide new methods for the prevention of tick-borne disease infections. Moreover, research of (but not limited to) tick AMPs could provide new ways to fight bacterial, viral, and protistan infections in the current dawn of the era of antibiotic-resistant bacteria.

acknowledgments This work was supported by Grant Nos. MSM 6007665801 and LC06009 (Ministry of Education), institutional research grants Z60220518 and KJB600960906 (Grant Agency of the ASCR), Grant Nos. 206/09/1782 and P302/11/1901 (Czech Science Foundation), and Grant Nos. CZB12963-CB-09 (M.G.) and CZB1-2966-CB-09 (N.R.) from the CRDF.

ref erences cited Araman, S.F. (1979) Protein digestion and synthesis in Ixodid females. In J. Rodriguez (Ed.), Recent Advances in Acarology, Vol. 1. New York: Academic Press, 385–395. Armstrong, P.B. (2001) The contribution of proteinase inhibitor to immune defense. Trends Immunol. 22:47–52. Balashov, Y.S. (1972) Bloodsucking ticks (Ixodidae)—vectors of diseases of man and animals. Misc. Pub. Entomol. Soc. Am. 8:161–376. Binnington, K.C. and Obenchain, F.D. (1982) Structure and function of the circulatory, nervous, and neuroendocrine systems of ticks. In F.D. Obenchain and R. Galun (Eds.), Physiology of Ticks. Oxford, UK: Pergamon Press, 277–350. Boldbaatar, D., Umemiya-Shirafuji, R., Liao, M., Tanaka, T., Xuan, X., and Fujisaki, K. (2010) Multiple vitellogenins from the Haemaphysalis longicornis tick are crucial for ovarian development. J. Insect Physiol. 56:1587–1598. Borovickova, B. and Hypsa, V. (2005) Ontogeny of tick hemocytes: a comparative analysis of Ixodes ricinus and Ornithodoros moubata. Exp. Appl. Acarol. 35:317–333. Braz, G.R., Coelho, H.S., Masuda, H., and Oliveira, P.L. (1999) A missing metabolic pathway in the cattle tick Boophilus microplus. Curr. Biol. 9:703–706. Brinton, L.P. and Burgdorfer, W. (1971) Fine structure of normal hemocytes in Dermacentor andersoni Stiles (Acari: Ixodidae). J. Parasitol. 57:1110–1127. Bulet, P., Stocklin, R., and Menin, L. (2004) Anti-microbial peptides: from invertebrates to vertebrates. Immunol. Rev. 198:169–184. Buresova, V., Franta, Z., and Kopacek, P. (2006) A comparison of Chryseobacterium indologenes pathogenicity to the soft tick Ornithodoros moubata and hard tick Ixodes ricinus. J. Invertebr. Pathol. 3:96–104. Buresova, V., Hajdusek, O., Franta, Z., Sojka, D., and Kopacek, P. (2009) IrAM—an α2-macroglobulin from the hard tick Ixodes ricinus: characterization and function in phagocytosis of a potential pathogen Cryseobacterium indologene. Dev. Comp. Immunol. 33:489–498. Burggren, W., Pinder, A., McMahon, B., Doyle, M., and Wheatly, M. (1990) Heart rate and hemolymph pressure responses to hemolymph volume changes in the land crab Cardisoma guanhumi: evidence for “Baroreflex” regulation. Physiol. Zool. 63:167–181. Carneiro, M.E. and Daemon, E. (2002) Comparative study of the hemolymph aspect from a few ticks species (Acari, Ixodidae): description of the hemocyte variation of adults of Amblyomma cajennense (Fabricius) Koch. Revta. Bras. Zool. 19 Suppl. 1:171–175. Ceraul, S.M. (2005) Defensin in the ticks Dermacentor variabilis and Ixodes scapularis. PhD thesis. Department of Biological Sciences, Old Dominion University, Norfolk, VA.


280

2 April 2013 5:34 PM


Circulatory System and Hemolymph 281 Ceraul, S.M., Dreher-Lesnick, S.M., Gillespie, J.J., Rahman, M.S., and Azad, A.F. (2007) New tick defensin isoform and antimicrobial gene expression in response to Rickettsia montanensis challenge. Infect. Immun. 75:1973–1983. Ceraul, S.M., Sonenshine, D.E., and Hynes, W.L. (2002) Resistance of the tick Dermacentor variabilis (Acari: Ixodidae) following challenge with the bacterium Escherichia coli (Enterobacteriales: Enterobacteriaceae). J. Med. Entomol. 39:376–383. Chinzei, Y. (1983) Quantitative changes of vitellogenin and vitellin in adult female ticks, Ornithodoros moubata, during vitellogenesis. Mie. Med. 32:117–127. Chinzei, Y. and Yano, I. (1985) Fat body is the site of vitellogenin synthesis in the soft tick, Ornithodoros moubata. J. Comp. Physiol. 155:671–678. Chrudimska, T., Chrudimsky, T., Golovchenko, M., Rudenko, N., and Grubhoffer, L. (2010) New defensins from hard and soft ticks: similarities, differences and phylogenetic analyses. Vet. Parasitol. 167:298–303. Cook, D., Stolts, D.B., and Pauley, C. (1985) Purification and preliminary characterization of insect spherulocytes. Insect Biochem. 15:419–426. Donohue, K.V., Khalil, S.M.S., Mitchell, R.D., Sonenshine, D.E., and Roe, R.M. (2008) Molecular characterization of the major hemelipoglycoprotein in ixodid ticks. Insect. Mol. Biol. 17:197–208. Dupejova, J., Sterba, J., Vancova, M., and Grubhoffer, L. (2011) Hemelipoglycoprotein from the ornate sheep tick, Dermacentor marginatus: structural and functional characterization. Parasit. Vectors 4:4. Eggenberger, L.R., Lamoreaux, W.J., and Coons, L.B. (1990) Hemocytic encapsulation of implants in the tick Dermacentor variabilis. Exp. Appl. Acarol. 9:279–287. El Shoura, S.M. (1986) Fine structure of the hemocytes and nephrocytes of Argas (Persicargas) arboreus (Ixodidae: Argasidae). J. Morphol. 189:17–24. El Shoura, S.M. (1989) Ultrastructure of the larval hemocytes and nephrocytes in the tick Ornithodoros (Pavlovskyella) erraticus (Ixodidae, Argasidae). Acarologia 30:35–40. Fogaca, A.C., Almeida, I.C., Eberlin, M.N., Tanaka, A.S., Bulet, P., and Daffre, S. (2006) Ixodidin, a novel antimicrobial peptide from the hemocytes of the cattle tick Boophilus microplus with inhibitory activity against serine proteinases. Peptides 27:667–674. Fogaca, A.C., da Silva, P.I., Jr., Miranda, M.T., Bianchi, A.G., Miranda, A., Ribolla, P.E., and Daffre, S. (1999) Antimicrobial activity of a bovine hemoglobin fragment in the tick Boophilus microplus. J. Biol. Chem. 274:25330–25334. Fogaca, A.C., Lorenzini, D.M., Kaku, L.M., Esteves, E., Bulet, P., and Daffre, S. (2004) Cysteine-rich antimicrobial peptides of the cattle tick Boophilus microplus: isolation, structural characterization and tissue expression profile. Dev. Comp. Immunol. 28:191–200. Ganz, T. (2003) Defensins: antimicrobial peptides of innate immunity. Nat. Rev. Immunol. 3:710–720. Gillespie, J.P., Kanost, M.R., and Trenczek, T. (1997) Biological mediators of insect immunity. Annu. Rev. Entomol. 42:611–643. Graça-Souza, A.V., Maya-Monteiro, C.M., Paiva-Silva, G.O., Braz, G.R.C., Paes, M.C., Sorgine, M.H.F., Oliveira, M.F., and Oliveira, P.L. (2006) Adaptations against heme toxicity in blood-feeding arthropods. Insect Biochem. Mol. Biol. 36:322–335. Granjeno-Colin, G., Hernandez-Ortiz, R., Mosqueda, J., Estrada-Mondaca, S., Figueroa, J.V., and Garcia-Vazquez, Z. (2008) Characterization of a vitellogenin gene fragment in Boophilus microplus ticks. Ann. N. Y. Acad. Sci. 1149:58–61. Grubhoffer, L., Golovchenko, M., Vancova, M., Zacharovova-Slavickova, K., Rudenko, N., and Oliver, J.H., Jr. (2005) Lyme borreliosis: insights into tick-host-borrelia interactions. Folia Parasitol. 52:279–294. Grubhoffer, L., Hypsa, V., and Volf, P. (1997) Lectins (hemagglutinins) in the gut of the important disease vector. Parasite 4:203–216. Grubhoffer, L. and Kovar, V. (1998) Arthropod lectins: affinity approaches in analysis and reparation of carbohydrate binding proteins. In A. Wiesner, G.B. Dunphy, V.J. Marmaras, I. Morishima, M. Sugumara, and M. Yamakava (Eds.), Techniques in Insect Immunology FITC-5. New York: SOS Publications, 47–57.


281

2 April 2013 5:34 PM


282 BIOLOGY OF TICKS Grubhoffer, L., Veres, J., and Dusbabek, F. (1991) Lectins as the molecular factors of recognition and defense reaction of ticks. In F. Dusbabek and V. Bukva (Eds.), Modern Acarology, Vol. 2. Prague: Academia, 381–388. Grunclova, L., Fouquier, H., Hypsa, V., and Kopacek, P. (2003) Lysozyme from the gut of the soft tick Ornithodoros moubata: the sequence, phylogeny and post-feeding regulation. Dev. Comp. Immunol. 27:651–660. Guddera, N.P., Neese, P.A., Sonenshine, D.E., Apperson, C.S., and Roe, R.M. (2001) Developmental profile, isolation, and biochemical characterization of a novel lipoglycoheme-carrier protein from the American dog tick, Dermacentor variabilis (Acari: Ixodidae) and observations on a similar protein in the soft tick, Ornithodoros parkeri (Acari: Argasidae). Insect Biochem. Mol. Biol. 31:299–311. Gudderra, N.P., Sonenshine, D.E., Apperson, C.S., and Roe, R.M. (2002) Hemolymph proteins in ticks. J. Insect Physiol. 48:269–278. Gullan, P.J. and Cranston, P.S. (2010) The Insects: An Outline of Entomology, 4th ed. Oxford, UK: Wiley-Blackwell. Habeeb, S.M. and El-Hag, H.A.A. (2008) Ultrastructural changes in hemocyte cells of hard tick (Hyalomma dromedarii: Ixodidae): a model of Bacillus thurigiensis var. thurigiensis H14*-endotoxin mode of action. Am.-Euras. J. Agric. Environ. Sci. 3:829–836. Hajdusek, O., Almazan, C., Loosova, G., Villar, M., Canales, M., Grubhoffer, L., Kopacek, P., and de la Fuente, J. (2010) Characterization of ferritin 2 for the control of tick infestations. Vaccine 28:2993–2998. Hajjar, N.P. (1972) Biochemical and physiological studies of certain ticks (Ixodidea). Fatty acid composition of lipids and free fatty acid fractions of hemolymph and gut and molting fluids of nymphal and female Hyalomma (H.) dromedarii Koch and H. (H.) anatolicum excavatum Koch (Ixodidae). J. Med. Entomol. 20:551–557. Hamdy, B.H., Taha, A.A., and Sidrak, W. (1974) Haemolymph and egg pigment of Dermacentor andersonii (Ixodidae) with reference to δ-aminolaevulic acid catabolism. Insect Biochem. 4:205–213. Hayes, J.D. and Strange, R.C. (2000) Glutathione S-transferase polymorphisms and their biological consequences. Pharmacology 61:154–166. Hefnawy, T. (1972a) Biochemical and physiological studies of certain ticks (Ixodidae). Hemolymph volume determined by isotope and dye dilution during the gonotrophic cycle of Argas (Persicargas) persicus (Oken) and A. (P.) arboreus Kaiser, Hoogstral, and Kohls (Argasidae). J. Parasitol. 58:358–364. Hefnawy, T. (1972b) Biochemical and physiological studies of certain ticks (Ixodidea). Osmotic pressure of hemolymph and gut and coxal fluids during the gonotrophic cycle of Argas (Persicargas) persicus (Oken) and A. (P.) arboreus Kaiser, Hoogstraal, and Kohls (Argasidae). J. Parasitol. 58:1197–1200. Hetru, C., Hoffmann, D., and Bullet, P. (1997) Antimicrobial peptides from insects. In P.T. Brey and D. Hultmark (Eds.), Molecular Mechanisms of Immune Responses in Insects. London: Chapman & Hall, 40–66. Horackova, J., Rudenko, N., Golovchenko, M., and Grubhoffer, L. (2010a) Der-p2 (Dermatophagoides pteronyssinus) allergen-like protein from the hard tick Ixodes ricinus—a novel member of ML (MD-2-related lipid-recognition) domain protein family. Parasitol. 137:1139–1149. Horackova, J., Rudenko, N., Golovchenko, M., Havlikova, S., and Grubhoffer, L. (2010b) IrML—a gene encoding a new member of the ML protein family from the hard tick, Ixodes ricinus. J. Vector Ecol. 35:410–418. Huang, X., Tsuji, N., Miyoshi, T., Nakamura-Tsuruta, S., Hirabayashi, J., and Fujisaki, K. (2007) Molecular characterization and oligosaccharide-binding properties of a galectin from the argasid tick Ornithodoros moubata. Glycobiol. 17:313–323. Hwang, K.-L.H. (2006) Physiological diversity and temperature hardening in adult tick Dermacentor variabilis (Acari: Ixodidae). PhD thesis. Ohio State University, Columbus, OH. Hynes, W.L., Ceraul, S.M., Todd, S.M., Seguin, K.C., and Sonenshine, D.E. (2005) A defensin-like gene expressed in the black-legged tick, Ixodes scapularis. Med. Vet. Entomol. 19:339–344. Hynes, W.L., Stokes, M.M., Hensley, S.M., Todd, S.M., and Sonenshine, D.E. (2008) Using RNA interference to determine the role of varisin in the innate immune system of the hard tick Dermacentor variabilis (Acari: Ixodidae). Exp. Appl. Acarol. 46:7–15.


282

2 April 2013 5:34 PM


Circulatory System and Hemolymph 283 Inoue, N., Hanada, K., Tsuji, N., Igarashi, I., Nagasawa, H., Mikami, T., and Fujisaki, K. (2001) Characterization of phagocytic hemocytes in Ornithodoros moubata (Acari: Ixodidae). J. Med. Entomol. 38:514–519. James, A.M. and Oliver, J.H., Jr. (1997) Purification and partial characterization of vitellin from the black-legged tick, Ixodes scapularis. Insect Biochem. Mol. Biol. 27:639–649. Jiang, H. and Kanost, M.R. (2000) The clip-domain family of serine proteinases in arthropods. Insect Biochem. Mol. Biol. 30:95–105. Johns, R., Sonenshine, D.E., and Hynes, W.L. (1998) Control of bacterial infections in the hard tick Dermacentor variabilis (Acari: Ixodidae): evidence for the existence of antimicrobial proteins in tick hemolymph. J. Med. Entomol. 35:458–464. Johns, R., Sonenshine, D.E., and Hynes, W.L. (2000) Response of the tick Dermacentor variabilis (Acari: Ixodidae) to hemocoelic inoculation of Borrelia burgdorferi (Spirochetales). J. Med. Entomol. 37:265–270. Johns, R., Sonenshine, D.E., and Hynes, W.L. (2001) Identification of a defensin from the hemolymph of the American dog tick, Dermacentor variabilis. Insect Biochem. Mol. Biol. 31:857–865. Kanost, M.R. and Jiang, H. (1996) Protease inhibitors in invertebrate immunity. In K. Soderhall, S. Iwanaga, and G. Vanta (Eds.), New Directions in Invertebrate Immunology. Fair Haven, NJ: SOS Publications, 155–173. Kaufman, W.R. and Phillips, J.E. (1973) Ion and water balance in the Ixodid tick Dermacentor andersoni. I. Routes of ion and water excretion. J. Exp. Biol. 58:523–536. Kawabata, S. and Tsuda, R. (2002) Molecular basis of non-self recognition by the horseshoe crab tachylectins. Biochim. Biophys. Acta 1572:414–421. Khalil, S.M.S., Donohue, K.V., Thompson, D.M., Jeffers, L.A., Ananthapadmanaban, U., Sonenshine, D.E., Mitchell, R.D., and Roe, R.M. (2011) Full-length sequence, regulation and developmental studies of a second vitellogenin gene from the American dog tick, Dermacentor variabilis. J. Insect Physiol. 57:400–408. Kocan, K.M., De La Fuente, J., Manzano-Roman, R., Naranjo, V., Hynes, W.L., and Sonenshine, D.E. (2008) Silencing expression of the defensin, varisin, in male Dermacentor variabilis by RNA interference results in reduced Anaplasma marginale infections. Exp. Appl. Acarol. 46:17–28. Kopacek, P., Vogt, R., Jindrak, L., Weise, C., and Safarik, I. (1999) Purification and characterization of the lysozyme from the gut of the soft tick Ornithodoros moubata. Insect Biochem. Mol. Biol. 29:989–997. Kopacek, P., Weise, C., Saravanan, T., Vıtova, K., and Grubhoffer, L. (2000) Characterization of an αmacroglobulin-like glycoprotein isolated from the plasma of the soft tick Ornithodoros moubata. Eur. J. Biochem. 267:465–475. Kovar, V., Kopacek, P., and Grubhoffer, L. (2000) Isolation and characterization of Dorin M, a lectin from plasma of the soft tick Ornithodoros moubata. Insect Biochem. Mol. Biol. 30:195–205. Kuhn, K.H. (1996) Mitotic activity of the hemocyte in the tick Ixodes ricinus (Acari: Ixodidae). Parasitol. Res. 82:511–517. Kuhn, K.H. and Haug, T. (1994) Ultrastructural, cytochemical, and immunocytochemical characterization of haemocytes of the hard tick Ixodes ricinus (Acari: Chelicerata). Cell Tissue Res. 277:493–504. Kuhn, K.H., Rittig, M., Häupl, T., and Burmester, G.R. (1994) Haemocytes of the hard tick Ixodes ricinus express coiling phagocytosis of Borrelia burgdorferi. Dev. Comp. Immunol. 18:S115. Kuhn, K.H., Uhlir, J., and Grubhoffer, L. (1996) Ultrastructural localization of a sialic acid-specific hemolymph lectin in the hemocytes and other tissues of the hard tick Ixodes ricinus (Acari: Chelicerata). Parasitol. Res. 82:215–221. Lai, R., Lomas, L.O., Jonczy, J., Turner, P.C., and Rees, H.H. (2004a) Two novel non-cationic defensinlike antimicrobial peptides from haemolymph of the female tick, Amblyomma hebraeum. Biochem. J. 379:681–685. Lai, R., Takeuchi, H., Lomas, L.O., Jonczy, J., Rigden, D.J., Rees, H.H., and Turner, P.C. (2004b) A new type of antimicrobial protein with multiple histidines from the hard tick, Amblyomma hebraeum. FASEB J. 18:1447–1449. Levenbook, L., Boctor, F.N., and Fales, H.M. (1980) Biochemical studies of tick embryogenesis. Free sugars in adult haemolymph and during embryogenesis of Dermacentor andersoni. J. Insect Physiol. 26:381–383.


283

2 April 2013 5:34 PM


284 BIOLOGY OF TICKS Loosova, G., Jindrak, L., and Kopacek, P. (2001) Mortality caused by experimental infection with the yeast Candida haemulonii in the adults of Ornithodoros moubata (Acarina: Argasidae). Folia Parasitol. (Praha) 48:149–153. Lucien, J., Reiffenstein, R., Zbitnew, G., and Kaufman, W.R. (1995) γ-aminobutyric acid (GABA) and other amino acids in tissues of the tick, Amblyomma hebraeum (Acari: Ixodidae) throughout the feeding and reproductive periods. Exp. Appl. Acarol. 19:617–631. Matsuo, T., Cerruto, N.C.A., Taylor, D., and Fujisaki, K. (2007) Immunohistochemical examination of PDGF-AB, TGF-beta and their receptors in the hemocytes of a tick, Ornithodoros moubata (Acari: Argasidae). J. Vet. Med. Sci. 69:17–20. Matsushita, M. and Fujita, T. (2001) Ficolins and the lectin complement pathway. Immunol. Rev. 180:78–85. Matsuyama, K. and Natori, S. (1988) Molecular cloning of cDNA for sapecin and unique expression of the sapecin gene during the development of Sarcophaga peregrina. J. Biol. Chem. 263:17117–17121. Maya-Monteiro, C.M., Alves, L.R., Pinhal, N., Abdalla, D.S.P., and Oliveira, P.L. (2004) HeLp, a hemetransporting lipoprotein with an antioxidant role. Insect Biochem. Mol. Biol. 34:81–87. Mulenga, A., Sugimoto, C., Ingram, G., Ohashi, K., and Misao, O. (2001) Characterization of two cDNAs encoding serine proteases from the hard tick Haemaphysalis longicornis. Insect Biochem. Mol. Biol. 31:817–825. Mulenga, A., Sugimoto, C., and Onuma, M. (2000) Issues in tick vaccine development: identification and characterization of potential candidate vaccine antigens. Microbes Infect. 2:1353–1361. Mulenga, A., Tsuda, A., Onuma, M., and Sugimoto, C. (2003) Four serine proteinase inhibitors (serpine) from the brown ear tick, Rhipicephalus appendiculatus; cDNA cloning and preliminary characterization. Insect Biochem. Mol. Biol. 33:267–276. Mullins, D.E. (1985) Chemistry and physiology of the hemolymph. In G.A. Kerkut and L.I. Gilbert (Eds.), Comprehensive Insect Physiology, Biochemistry and Pharmacology, Vol. 3. Oxford, UK: Pergamon Press, 355–400. Nakajima, Y., Ishibashi, J., Yukuhiro, F., Asaoka, A., Taylor, D., and Yamakawa, M. (2003a) Antimicrobial activity and mode of action of tick defensin against Gram-positive bacteria. Biochim. Biophys. Acta 1624:125–130. Nakajima, Y., Saido-Sakanaka, H., Taylor, D., and Yamakawa, M. (2003b) Up-regulated humoral immune response in the soft ticks, Ornithodoros moubata (Acari: Argasidae). Parasitol. Res. 91:476–481. Nakajima, Y., van der Goes van Naters-Yasui, A., Taylor, D., and Yamakawa, M. (2001) Two isoforms of a member of the arthropod defensin family from the soft tick, Ornithodoros moubata (Acari: Argasidae). Insect Biochem. Mol. Biol. 31:747–751. Nakajima, Y., van der Goes van Naters-Yasui, A., Taylor, D., and Yamakawa, M. (2002) Antibacterial peptide defensin is involved in midgut immunity of the soft tick, Ornithodoros moubata. Insect Mol. Biol. 11:611–618. Obenchain, F.D. and Oliver, J.H., Jr. (1976) The heart and arterial circulatory system of ticks (Acari: Ixodioidea). J. Arachnol. 3:57–74. Pace, K.E., Lebestky, T., Hummel, T., Arnoux, P., Kwan, K., and Baum, L.G. (2002) Characterization of a novel Drosophila melanogaster galectin. Expression in developing immune, neural, and muscle tissues. J. Biol. Chem. 277:13091–13098. Pan, H.J., Teng, L.J., Chen, Y.C., Hsueh, P.R., Yang, P.C., Ho, S.W., and Luh, K.T. (2000) High protease activity of Chryseobacterium indologenes isolates associated with invasive infection. J. Microbiol. Immunol. Infect. 33:223–226. Pereira, L.S., Oliveira, P.L., Barja-Fidalgo, C., and Daffre, S. (2001) Production of reactive oxygen species by hemocytes from the cattle tick Boophilus microplus. Exp. Parasitol. 99:66–72. Podboronov, V.M. (1991) Antibacterial protective mechanisms of ixodoid ticks. In F. Dusbábek and F. bukva (Eds.), Modern Acarology, Vol. 2. Prague: Academia, 375-380. Podboronov, V.M. and Berdyev, A. (1991) Protective Mechanisms of Ixodid Ticks and Their Vertebrate Hosts (Host-Parasite Relationship). Ashgabad, Turkmenistan: Ylym (in Russian). Polanowski, A. and Wilusz, T. (1996) Serine proteinase inhibitors from insect hemolymph. Acta Biochim. Pol. 43:445–453.


284

2 April 2013 5:34 PM


Circulatory System and Hemolymph 285 Rego, R.O., Hajdusek, O., Kovar, V., Kopacek, P., Grubhoffer, L., and Hypsa, V. (2005) Molecular cloning and comparative analysis of fibrinogen-related proteins from the soft tick Ornithodoros moubata and the hard tick Ixodes ricinus. Insect Biochem. Mol. Biol. 35:991–1004. Rego, R.O.M. (2005) Identification and comparative analysis of innate immune proteins in ticks. Ph.D. thesis. Faculty of Science, University of South Bohemia, Ceske Budejovice, Czech Republic. Rittig, M.G., Kuhn, K.H., Dechant, C.A., Gauckler, A., Modolell, M., Ricciardi-Castagnoli, P., Krause, A., and Burmester, G.R. (1996) Phagocytes from both vertebrate and invertebrate species use “coiling” phagocytosis. Dev. Comp. Immunol. 20:393–406. Rudenko, N., Golovchenko, M., Edwards, M.J., and Grubhoffer, L. (2005) Differential expression of Ixodes ricinus tick genes induced by blood feeding or Borrelia burgdorferi infection. J. Med. Entomol. 42:36–41. Rudenko, N., Golovchenko, M., and Grubhoffer, L. (2007) Gene organization of a novel defensin of Ixodes ricinus: first annotation of an intron/exon structure in a hard tick defensin gene and first evidence of the occurrence of two isoforms of one member of the arthropod defensin family. Insect Mol. Biol. 16:501–507. Saito, Y., Konnai, S., Yamada, S., Imamura, S., Nishikado, H., Ito, T., Onuma, M., and Ohashi, K. (2009) Identification and characterization of antimicrobial peptide, defensin, in the taiga tick, Ixodes persulcatus. Insect Mol. Biol. 18:531–539. Schriefer, M.E. (1991) Vitellogenesis in Hyalomma dromedarii (Acari: Ixodidae): a model for analysis of endocrine regulation in ixodid ticks. Ph.D. thesis. Old Dominion University, Norfolk, VA. Silva, F.D., Rezende, C.A., Rossi, D.C., Esteves, E., Dyszy, F.H., Schreier, S., Gueiros-Filho, F., Campos, C.B., Pires, J.R., and Daffre, S. (2009) Structure and mode of action of microplusin, a copper II chelating antimicrobial peptide from the cattle tick Rhipicephalus (Boophilus) microplus. J. Biol. Chem. 284:34735–34746. Simser, J.A., Macaluso, K.R., Mulenga, A., and Azad, A.F. (2004a) Immune-responsive lysozymes from hemocytes of the American dog tick, Dermacentor variabilis and an embryonic cell line of the Rocky mountain wood tick, D. andersoni. Insect Biochem. Mol. Biol. 34:1235–1246. Simser, J.A., Mulenga, A., Macaluso, K.R., and Azad, A.F. (2004b) An immune responsive factor D-like serine proteinase homologue identified from the American dog tick, Dermacentor variabilis. Insect Mol. Biol. 13:25–35. Socolovschi, C., Mediannikov, O., Raoult, D., and Parola, P. (2009) The relationship between spotted fever group Rickettsiae and Ixodid ticks. Vet. Res. 40:34. Sonenshine, D.E. (1991) Biology of Ticks, Vol. 1. New York: Oxford University Press. Sonenshine, D.E., Ceraul, S.M., Hynes, W.E., Macaluso, K.R., and Azad, A.F. (2002) Expression of defensin-like peptides in tick hemolymph and midgut in response to challenge with Borrelia burgdorferi, Escherichia coli and Bacillus subtilis. Exp. Appl. Acarol. 28:127–134. Sonenshine, D.E. and Hynes, W.L. (2008) Molecular characterization and related aspects of the innate immune response in ticks. Front. Biosci. 13:7046–7063. Sonenshine, D.E., Hynes, W.L., Ceraul, S.M., Mitchell, R., and Benzine, T. (2005) Host blood proteins and peptides in the midgut of the tick Dermacentor variabilis (Say) contribute to bacterial control. Exp. Appl. Acarol. 36:207–223. Sterba, J., Dupejova, J., Fiser, M., Vancova, M., and Grubhoffer, L. (2011) Fibrinogen-related proteins in Ixodid ticks. Parasit. Vectors. 4:127. Stopforth, E., Albert, W.H., Neitz, A.W.H., and Gaspar, A.R.M. (2010) A proteomics approach for the analysis of hemolymph proteins involved in the immediate defense response of the soft tick, Ornithodoros savignyi, when challenged with Candida albicans. Exp. Appl. Acarol. 51:309–325. Tanaka, T., Kawano, S., Nakaoa, S., Umemiya-Shirafujia, R., Rahman, Md. M., Boldbaatar, D., Battur, B., Liao, M., and Fujisaki, K. (2010) The identification and characterization of lysozyme from the hard tick Haemaphysalis longicornis. Ticks Tick-borne Dis. 1:178–185. Taylor, D. (2006) Innate immunity in ticks: a review. J. Acarol. Soc. Japan 15:109–127. Taylor, D., Chinzei, Y., Miura, K., and Anado, K. (1991) Vitellogenin synthesis, processing and hormonal regulation in the tick, Ornithodoros parkeri (Acari: Argasidae). Insect Biochem. 21:723–733. Thompson, D.M., Khalil, S.M.S., Jeffers, L.A., Sonenshine, D.E., Mitchell, R.D., Osgood, C.J., and Roe, R.M. (2007) Sequence and the developmental and tissue-specific regulation of the first complete


285

2 April 2013 5:34 PM


286 BIOLOGY OF TICKS vitellogenin messenger RNA from ticks responsible for heme sequestration. Insect Biochem. Mol. Biol. 37:363–374. Todd, S.M., Sonenshine, D.E., and Hynes, W.L. (2007) Tissue and life-stage distribution of a defensin gene in the Lone Star tick, Amblyomma americanum. Med. Vet. Entomol. 21:141–147. Tsuji, N., Kamio, T., Isobe, T., and Fujisaki, K. (2001) Molecular characterization of a peroxiredoxin from the hard tick Haemaphysalis longicornis. Insect Mol. Biol. 10:121–129. Valenzuela, J.G., Francischetti, I.M., Pham, V.M., Garfield, M.K., Mather, T.N., and Ribeiro, J.M. (2002) Exploring the sialome of the tick Ixodes scapularis. J. Exp. Biol. 205:2843–2864. Vasta, G.R., Ahmed, H., Finl, N.E., Elola, M.T., Marsh, A.G., Snowden, A., and Odom, E.W. (1994) Animal lectins as self/non-self recognition molecules. Biochemical and genetic approaches to understanding their biological roles and evolution. Ann. N. Y. Acad. Sci. 712:55–73. Vasta, G.R., Quesenberry, M., Ahmed, H., and O’Leary, N. (1999) C-type lectins and galectins mediate innate and adaptive immune functions: their roles in the complement activation pathway. Dev. Comp. Immunol. 23:401–420. Vierstraete, E., Cerstiaens, A., Baggerman, G., Van den Bergh, G., De Loof, A., and Schoofs, L. (2003) Proteomics in Drosophila melanogaster: first 2D database of larval hemolymph proteins. Biochem. Biophys. Res. Commun. 304:831–838. Wang, X., Rocheleau, T.A., Fuchs, J.F., Hillyer, J.F., Chen, C.C., and Christensen, B.M. (2004) A novel lectin with a fibrinogen-like domain and its potential involvement in the innate immune response of Armigeres subalbatus against bacteria. Insect Mol. Biol. 13:273–282. Wu, M., Maier, E., Benz, R., and Hancock, R.E.W. (1999) Mechanism of interaction of different classes of cationic antimicrobial peptides with planar bilayers and with the cytoplasmic membrane of Escherichia coli. Biochem. 38:7235–7242. Zhioua, E., Lebrun, R.A., Johnson, P.W., and Ginsberg, H.S. (1996) Ultrastructure of the hemocytes of Ixodes scapularis (Acari: Ixodidae). Acarologia 37:173–179.


286

2 April 2013 5:34 PM