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Contents lists available at ScienceDirect

Ticks and Tick-borne Diseases

jo ur nal homepage: www.elsev ier .com/ locate / t tbd is

riginal article

ost-derived transferrin is maintained and transferred from midguto ovary in Haemaphysalis longicornis ticks

iroyuki Moria, Remil Linggatong Galaya,b, Hiroki Maedaa,b, Tomohide Matsuoc,ika Umemiya-Shirafujid, Masami Mochizukia,b, Kozo Fujisakie, Tetsuya Tanakaa,b,∗

Laboratory of Emerging Infectious Diseases, Joint Faculty of Veterinary Medicine, Kagoshima University, Korimoto, Kagoshima 890-0065, JapanDepartment of Pathological and Preventive Veterinary Science, The United Graduate School of Veterinary Science, Yamaguchi University, Yoshida,amaguchi 753-8515, JapanLaboratory of Parasitology, Joint Faculty of Veterinary Medicine, Kagoshima University, Korimoto, Kagoshima 890-0065, JapanNational Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555,

apanNational Agricultural and Food Research Organization, Kannondai, Tsukuba, Ibaraki 305-0856, Japan

r t i c l e i n f o

rticle history:eceived 12 April 2013eceived in revised form8 September 2013ccepted 19 September 2013vailable online xxx

eywords:aemaphysalis longicornis

a b s t r a c t

Transferrin is known to be an iron transporter in vertebrates and several arthropods. Iron from hostblood is essential for ovarian development in blood-sucking arthropods. However, tick transferrin hasbeen identified in only a few species, and its function has yet to be elucidated, resulting in incompleteunderstanding of iron metabolism in ticks. Here, we investigated the transfer of host-derived transferrinin the hard tick Haemaphysalis longicornis using immunological methods. Western blot showed that host-derived transferrin was maintained in all developmental stages of ticks up to 28 days after engorgementand was detected in the midgut and the ovary of adult females following blood feeding. However, nohost-derived transferrin was detected in eggs after laying or in larvae after hatching, indicating that host-

rotein transferronron-binding proteinransferrin

derived transferrin is not transferred to offspring transovarially. Indirect immunofluorescent antibodytesting showed the localization of host-derived transferrin in digestive cells of the midgut and oocytes ofthe ovary from engorged adult females. These results suggest that host-derived transferrin is transferredto the ovary through the midgut and the hemolymph, and raise the possibility of the function of host-derived transferrin as an iron source in the ovary, providing additional insight on iron metabolism inticks.

ntroduction

The only food source for ticks at all developmental stages isertebrate blood. Therefore, ticks have a very unique lifestyle andompletely depend on vertebrate blood for their survival, devel-pment, and egg production. Moreover, they ingest large amountsf blood in a meal, inducing a remarkable increase, especially inhe weight of adult females (Romoser, 1996). Many host bloodomponents are taken up during blood feeding. Blood digestionn ticks occurs intracellularly within the endo-lysosomal vesicles

Please cite this article in press as: Mori, H., et al., Host-derived transferrin ilongicornis ticks. Ticks Tick-borne Dis. (2013), http://dx.doi.org/10.1016/j.t

r the cytosol of midgut cells (Sonenshine, 1991; Horn et al., 2009;ojka et al., 2013). Hemoglobin, the most abundant iron-bindingrotein in mammalian blood, is known to be digested by means of

∗ Corresponding author at: Laboratory of Emerging Infectious Diseases, Joint Fac-lty of Veterinary Medicine, Kagoshima University, 1-21-24 Korimoto, Kagoshima90-0065, Japan. Tel.: +81 99 285 3570; fax: +81 99 285 3570.

E-mail address: tetsuya@ms.kagoshima-u.ac.jp (T. Tanaka).

877-959X/$ – see front matter © 2013 Elsevier GmbH. All rights reserved.ttp://dx.doi.org/10.1016/j.ttbdis.2013.09.004

© 2013 Elsevier GmbH. All rights reserved.

proteolytic enzymes in the digestive cells of midguts, resulting inthe generation of large amounts of heme (Tarnowski and Coons,1989; Gough and Kemp, 1995; Mendiola et al., 1996) and free aminoacids (Sojka et al., 2013).

Iron is an essential molecule of both vertebrates and arthropods.It plays an important role as a cofactor of proteins in various phys-iological processes, such as oxygen metabolism, the tricarboxylicacid cycle, steroid production, gene regulation, amino acid produc-tion, and DNA biosynthesis (Ong et al., 2006; Andrews, 2008). Inaddition, some arthropods require iron for optimal egg develop-ment and viable offspring (Geiser and Winzerling, 2012). However,iron can be also toxic for them, because it leads to the productionof dangerous reactive oxygen species and the generation of highlyreactive radicals (Wang and Pantopoulos, 2011). These radicalshave potential for damaging cellular macromolecules and promote

s maintained and transferred from midgut to ovary in Haemaphysalistbdis.2013.09.004

cell death and tissue injury (Papanikolaou and Pantopoulos, 2005).Thus, both vertebrates and arthropods evolved various specializedproteins, such as transferrin or ferritin, for binding iron to keepit in a safe and bioavailable form (Arosio et al., 2009). Vertebrate

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ransferrins have been well characterized and are known to be anntercellular iron transporter for cell proliferation, differentiation,nd organogenesis. Meanwhile, arthropod transferrins have beenlso identified and characterized in several insect families (Hueberst al., 1988; Jamroz et al., 1993; Yun et al., 1999; Yoshiga et al., 1997,999) and were claimed to function in iron transport and innate

mmunity (Harizanova et al., 2005; Guz et al., 2007; Zhou et al.,009). Arthropod transferrin also acts as a vitellogenin protein or a

uvenile hormone-regulated protein (Nichol et al., 2002; Guz et al.,007).

Vertebrate iron-binding proteins in blood mainly consist oferum transferrin or hemoglobin (Winzerling and Pham, 2006),o blood-sucking arthropods have to balance these molecules totilize iron or prevent iron toxicity. It has been found that theattle tick Rhipicephalus (Boophilus) microplus lacks a functionaleme synthesis pathway (Braz et al., 1999), and thus heme released

rom host hemoglobin is sequestered by hemelipoglyco-carrierroteins in the American dog tick Dermacentor variabilis and theoft tick Ornithodoros parkeri (Gudderra et al., 2001), or by theeme lipoprotein (HeLp) in R. (B.) microplus (Maya-Monteiro et al.,000), and transported to the hemolymph and peripheral tissuesy these proteins. Heme is also found to be accumulated in apecialized organelle called hemosome as part of a detoxificationechanism (Lara et al., 2003). Meanwhile, the mechanism of non-

eme iron traffic is still poorly understood in ticks. Transferrinas been identified in only a few tick species, including the black-

egged tick Ixodes scapularis (GenBank: EEC13740) and the zebraick Rhipicephalus (Boophilus) pulchellus (GenBank: JAA63555), buthe function has not been fully characterized. This fact leaveshe knowledge of tick iron metabolism incomplete, compared tohat of other blood-sucking arthropods (Zhou et al., 2007; Phamnd Winzerling, 2010). In the present study, we investigated theransfer of host-derived transferrin in the hard tick Haemaphysalisongicornis using immunoblot and immunohistological analyses.

aterials and methods

icks and experimental animals

The parthenogenetic Okayama strain of H. longicornis, includ-ng larvae, nymphs, and adult females, maintained by feeding onapanese white rabbits (Kyudo, Kumamoto, Japan) in our labora-ory as described previously (Fujisaki, 1978), was used throughouthis study. All ticks used had been kept at 15 ◦C and 85% rel-tive humidity for 3 months after their previous blood feedingr hatching. Rabbit and female ICR mouse (Kyudo) care followedpproved guidelines from the Animal Care and Use Commit-ee of Kagoshima University (Approval number A08010). Ticksn most experiments were put onto the ears of rabbits and col-ected during blood feeding (partially fed) or upon engorgement.n experiments using mice, 2 adult tick females were put ontohe shaven back of each mouse and collected upon engorge-

ent. The collected ticks were maintained at 25 ◦C and 85%elative humidity to keep their physiological processes until user to allow engorged adult females to lay eggs. Laid eggs andatched larvae were maintained under the same conditions untilse.

ick organ dissection and hemolymph extraction

The salivary glands, midgut, fat body, ovary, and synganglion of

Please cite this article in press as: Mori, H., et al., Host-derived transferrin ilongicornis ticks. Ticks Tick-borne Dis. (2013), http://dx.doi.org/10.1016/j.t

oth partially fed and fully engorged adult females were dissectedut. These dissected organs were thoroughly rinsed in phos-hate buffered saline (PBS) to remove other tissue remnants. Theemolymph from partially fed and fully engorged adult females

PRESS Diseases xxx (2013) xxx– xxx

was directly collected from tick legs, amputated at the coxa-trochanter joint, using a capillary tube with PBS. Dissection andhemolymph collection from ticks were performed under the dis-secting microscope. Samples were placed in individual tubes andstored at −30 ◦C until use.

Protein extraction

The extraction of proteins from whole ticks at different devel-opmental stages, including laid eggs and hatched larvae, or fromthe dissected organs was performed as described previously(Boldbaatar et al., 2006; Aung et al., 2011). Briefly, the sampleswere transferred to new tubes, homogenized by Automill (Tokken,Chiba, Japan) or by sterilized plastic bars in the case of the dissectedorgans, and rotated for mixing overnight at 4 ◦C with a proteinaseinhibitor (Complete Mini; Roche Diagnostics, Basel, Switzerland) inPBS. Subsequently, the samples were centrifuged at 20,000 × g for10 min at 4 ◦C, and then the supernatants were recovered. The col-lected hemolymph was diluted with PBS to a final volume of 25 �leach. All the protein extracts were stored at 4 ◦C until use.

Western blot analysis

The concentration of the protein extracts was determined usingMicro BCATM Protein Assay Kit (Thermo Fisher Scientific, MA, USA).The protein extracts (8 �g from whole ticks, 12 �g from organs,eggs, hatched larvae, whole rabbit-fed and mouse-fed ticks, and16 �g from hemolymph) were subjected to electrophoresis in anSDS-polyacrylamide gel and were transferred onto a PVDF mem-brane (Millipore, MA, USA). After blocking with 3% skim milk inPBS containing 0.05% Tween 20 (0.05% PBS-T) overnight at 4 ◦C,the membrane was incubated with sheep anti-rabbit transferrinIgG (Bethyl Laboratories, TX, USA) as the first antibody, dilutedto 1:1000 with 3% skim milk in 0.05% PBS-T, for 1 h at 37 ◦C. Themembrane was washed with 0.05% PBS-T three times and thenincubated with donkey anti-sheep IgG conjugated with horseradishperoxidase (HRP) (R&D Systems, MN, USA) as the second antibody,diluted to 1:1000 with 3% skim milk in 0.05% PBS-T, for 1 h at37 ◦C. For detection of mouse-derived transferrin, the same methodwas carried out apart from using antibodies consisting of rabbit-derived Anti-Transferrin antibody (ab1223; Abcam, Cambridge,UK) for mouse transferrin as the first antibody and HRP-conjugateddonkey-derived ECLTM Anti-rabbit IgG (GE Healthcare, Uppsala,Sweden) as the second antibody. Specific protein bands werevisualized using 3,3′-diaminobenzidine tetrahydrochloride (DAB)staining.

Indirect immunofluorescent antibody test (IFAT)

IFAT was performed to investigate the localization of rabbit-derived transferrin in H. longicornis organs, according to the methodpreviously described (Boldbaatar et al., 2006; Aung et al., 2011).Briefly, the midguts and ovaries dissected from engorged adultfemales, as described in the preceding section, were fixed overnightin 4% paraformaldehyde in PBS containing 0.1% glutaraldehyde.After a series of washings with different concentrations of sucrosein the PBS solution, the organs were embedded in Tissue-Tek® O.C.Tcompound (Sakura Finetek Japan, Tokyo, Japan) and frozen. Thefrozen sections were cut 10 �m thick using a cryostat (Leica CM3050; Leica Microsystems, Wetzlar, Germany) and were mountedon MAS-coated glass slides (Matsunami Glass, Osaka, Japan). Afterwashing with PBS followed by blocking with 5% skim milk in PBS

s maintained and transferred from midgut to ovary in Haemaphysalistbdis.2013.09.004

overnight at 4 ◦C, sheep anti-rabbit transferrin IgG (Bethyl Labo-ratories; 1:1000 dilution with 5% skim milk in PBS) was appliedas the first antibody and incubated overnight at 4 ◦C or for 2 h atroom temperature. Sheep IgG (Sigma–Aldrich, MO, USA; 1:1000

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Fig. 1. Detection of rabbit-derived transferrin in whole bodies of engorged H. longi-cornis ticks including larvae, nymphs, and adult females. The protein extracts (8 �geach) were loaded in each lane, and then Western blot analysis was performed usingsheep anti-rabbit transferrin IgG as the first antibody and HRP-conjugated donkeyanti-sheep IgG as the second antibody. Bands were visualized through DAB stain-ilu

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Fig. 2. Detection of rabbit-derived transferrin in the hemolymph (A) and in differentorgans (B) of engorged adult female H. longicornis. The protein extracts (12 �g fromorgans, 16 �g from hemolymph) were loaded in each lane, and then Western blotanalysis was performed using sheep anti-rabbit transferrin IgG as the first antibodyand HRP-conjugated donkey anti-sheep IgG as the second antibody. Twelve �g of

ng. Twelve �g of total protein from rabbit blood and from rabbit transferrin wereoaded as positive controls. The numbers represent the days after engorgement. Uf,nfed ticks; RB, rabbit blood; RTF, rabbit transferrin.

ilution with 5% skim milk in PBS) was applied as a negative con-rol instead of the sheep anti-rabbit transferrin IgG. After washingith PBS 3 times, donkey anti-sheep IgG conjugated with Alexa

luor 488 (Life Technologies, CA, USA; 1:1000 dilution with 5%kim milk in PBS) was applied subsequently as the second anti-ody and incubated for 1 h at room temperature. The sectionsere mounted in Vectashield® with 4′,6-diamino-2-phenylindoleounting medium (Vector Laboratories, CA, USA), observed under

fluorescence microscope (IX71; Olympus, Tokyo, Japan) mountedith a DP71 camera, and processed using DP Controller software

Olympus).

esults

etection of rabbit-derived transferrin in different developmentaltages

First of all, the specificity of the first antibody (sheep anti-rabbitransferrin IgG) was checked using engorged ticks fed on rabbit or

ouse, mouse blood, rabbit blood, bovine transferrin, and rabbitransferrin. As shown in Fig. S1, the first antibody detected onlyabbit transferrin (approximately 77 kDa), thus, its specificity wasalidated.

To determine whether rabbit-derived transferrin is maintainedn larvae, nymphs, and female adults after engorgement and toheck the duration of the maintenance, Western blot analysis waserformed. Both larvae and adult females showed a similar patternf the specific bands when measured against rabbit-derived trans-errin throughout the observed days (Fig. 1). However, a decrease inand intensity was observed in nymphs from day 14 after engorge-ent when their molting had been definitely completed. Moreover,

he specific bands of rabbit-derived transferrin were also observedn both unfed nymphs and adult females, implying that rabbit-erived transferrin was retained even after 3 months had passedrom their previous blood meal. In conjunction with this result,

ouse-derived transferrin (approximately 78 kDa) was detectedrom mouse-fed engorged adult females (Fig. S3A), which furtherupports the suggestion that serum transferrin from host blood-tream could be maintained in the tick body.

etection of rabbit-derived transferrin in the hemolymph andarious organs of engorged adult females

Since the maintenance of rabbit-derived transferrin in whole

Please cite this article in press as: Mori, H., et al., Host-derived transferrin ilongicornis ticks. Ticks Tick-borne Dis. (2013), http://dx.doi.org/10.1016/j.t

. longicornis ticks was confirmed (Fig. 1), we next tried to detectabbit-derived transferrin from the hemolymph and various organsf engorged adult females through Western blot analysis. Rabbit-erived transferrin was detected in the hemolymph on days 1 and

total protein from rabbit blood and from rabbit transferrin were loaded as positivecontrols. The numbers represent the days after engorgement. Uf, unfed ticks; RB,rabbit blood; RTF, rabbit transferrin.

7 after engorgement (Fig. 2A) despite the failure to detect it dur-ing blood feeding (Fig. S2A). In various organs, specific bands ofrabbit-derived transferrin were detected in the midgut and theovary (Fig. 2B), and interestingly, this persisted for up to day 28after engorgement, in accordance with the results obtained fromwhole adult female ticks (Fig. 1). The midgut and the ovary sam-ples showed rabbit-derived transferrin even during blood feeding(Fig. S2B). These results raise the possibility that rabbit-derivedtransferrin might be transferred to the hemolymph and the ovaryfrom the midgut. Among the organs of unfed adult females, onlythe ovary showed rabbit-derived transferrin, suggesting that theremaining rabbit-derived transferrin acquired from the nymphalblood meal was aggregated mainly in the ovary. To support theseresults, we attempted to check the hemolymph and organs ofmouse-fed engorged adult females (Fig. S3B). Even though constantdetection was not gained, mouse-derived transferrin was detectedafter engorgement in the hemolymph, midgut, and ovary, con-firming that host-derived transferrin could be transferred to thehemolymph and aggregated in the ovary. Meanwhile, the otherfirst antibody, sheep anti-rabbit transferrin IgG, did not react withmouse-derived transferrin (Fig. S3C).

We tried also to detect rabbit-derived transferrin from laid eggsand from hatched larvae with the hypothesis that host-derivedtransferrin could be transferred transovarially. However, rabbit-derived transferrin was not detected in the eggs or larvae at anyday of development (Fig. S4).

Localization of rabbit-derived transferrin in the midgut and ovaryof engorged adult females

To investigate the localization of rabbit-derived transferrin inthe midgut and ovary of engorged adult H. longicornis females, IFAT

s maintained and transferred from midgut to ovary in Haemaphysalistbdis.2013.09.004

was performed on days 1 and 7 after engorgement (Fig. 3). In themidgut, strong fluorescence was found from the cytoplasm of diges-tive cells on both days, which was prominent near the basal lamina(Fig. 3A). In the ovary, rabbit-derived transferrin showed strong

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Fig. 3. Localization of rabbit-derived transferrin in the midgut (A) and the ovary (B) of unfed and engorged adult female H. longicornis. IFAT was performed using sheepanti-rabbit transferrin IgG as the first antibody and donkey anti-sheep IgG conjugated with Alexa 488 as the second antibody (Anti-TF panels). For control sections, sheep IgGwas used instead of sheep anti-rabbit transferrin IgG (Control panels). The nucleus was stained with blue fluorescence of DAPI. DAPI and Alexa represent sections stainedwith DAPI (blue) and donkey anti-sheep IgG conjugated with Alexa 488 (green), respectively. The overlaid images are shown in the rightmost panels. Each arrow showsp engorgB e read

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oints to distinct positive green fluorescence. The numbers represent the days after

ars = 100 �m. (For interpretation of the references to color in this figure legend, th

uorescence in the cytoplasm of oocytes on both days (Fig. 3B).owever, no fluorescence was found in the oviducts. The notableifference between the results of midgut and ovary sections washat fluorescence in the ovary was also present in unfed adultemales, which was in accordance with the result of Western blotnalysis (Fig. 2). Only very weak fluorescence was detected fromhe control sections of both organs. The fat body showed no fluo-escence to rabbit-derived transferrin (data not shown).

iscussion

Iron plays a critical role in many physiological processes, both inertebrates and arthropods. Transferrin is related to iron transportn mammals (Aisen, 1998) and is also considered to be an iron-inding protein in several insects (Geiser and Winzerling, 2012).owever, only a few tick transferrins are reported so far, and ironetabolism in ticks is poorly understood. In this study, we aimed to

nvestigate the transfer of host-derived transferrin in H. longicor-is to clarify its importance in the tick. Haemaphysalis longicornishowed a characteristic pattern of maintenance of rabbit-derivedransferrin in all developmental stages. This result indicates that H.ongicornis is capable of retaining host-derived transferrin through-ut each developmental stage. The specificity of the first antibodysed in Western blotting and IFAT was verified because no cross-eactivity was detected from ticks fed on mice, mouse blood, or

Please cite this article in press as: Mori, H., et al., Host-derived transferrin ilongicornis ticks. Ticks Tick-borne Dis. (2013), http://dx.doi.org/10.1016/j.t

ovine transferrin. In addition, compared to tick transferrin iden-ified from I. scapularis and R. (B.) pulchellus, which shares around0% identity with rabbit transferrin, mouse and bovine transferrinhare higher identities of 73% and 69%, respectively. Thus, our result

ement. Uf, unfed adult females; L, lumen; DC, digestive cell; O, oocyte; OD, oviduct.er is referred to the web version of the article.)

further indicates that there is a very high possibility that the firstantibody did not detect tick transferrin.

In the course of studies on host proteins in ticks, Ackermanet al. (1981) reported that host-derived transferrin appeared in thehemolymph of D. variabilis after blood feeding. Wickramasekaraet al. (2008) also detected host-derived transferrin in wholenymphal ticks of the lone-star tick Amblyomma americanum 3months post molt. However, these studies did not investigate thetransfer of host-derived transferrin in tick organs. To our knowl-edge, this is the first report demonstrating the movement ofhost-derived transferrin within ticks, particularly from the midgutto the ovary via the hemolymph. This new finding is further vali-dated by our additional result showing mouse-derived transferrinin the hemolymph and the ovary of mouse-fed engorged adult H.longicornis females with no band in organs from the unfed adultfemale, which means that transferrin from the tick’s most recentblood meal can be transferred to the ovary through the midgutand the hemolymph. The results by IFAT further showed the lineardistribution of host-derived transferrin along the basal lamina ofdigestive cells and the accumulation in the cytoplasm of oocytes,indicating that host-derived transferrin passed through digestivecells of the midgut and was then incorporated into oocytes via thehemolymph after blood feeding.

Female ixodid ticks have a very high fecundity; they are capableof laying thousands of eggs in a single batch. This high fecun-dity relies on essential nutrients, including iron from vertebrate

s maintained and transferred from midgut to ovary in Haemaphysalistbdis.2013.09.004

blood (Braz et al., 1999). A majority of iron in the host blood ispresent as ferric transferrin and hemoglobin (Winzerling and Pham,2006), and thus, the iron required by ticks might come from hosthemoglobin or transferrin. The movement of heme-iron derived

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rom host blood has been more investigated than that of non-hemeron in ticks. In adult R. (B.) microplus females, the role of HeLp as aransporter of heme-iron was demonstrated, with increased HeLpetected in the ovary where heme- or non-heme iron is importantor normal oocyte growth (vitellogenesis) (Maya-Monteiro et al.,000). The movement of host-derived non-heme iron has been

ust hypothesized. Nevertheless, the study and the proposal on theovement of non-heme iron by Hajdusek et al. (2009) supported

he general model of iron metabolism in tick physiology. Accordingo them, host ferric-transferrin may play the most important role as

source of non-heme iron. However, they also suggested that host-erived transferrin is degraded in the endosomes of the digestiveells. In our present study, host-derived transferrin was found in theidgut, the hemolymph, and the ovary without degradation. Thus,

he results of our study provide a new insight into their putativeodel of iron metabolism in ticks. In mosquitoes, host-transferrin

ron is more bioavailable and thus more utilized (Zhou et al., 2007).aken together, our study raises the possibility that host-derivedransferrin might play a role as an iron source and as a transporter,specially to the ovary.

Ferritin genes have been identified from 8 species of hard ticksXu et al., 2004) and soft ticks (Kopácek et al., 2003). Althougherritin plays a main role in intracellular iron storage in verte-rates, it is also involved in iron transport in arthropods (Phamnd Winzerling, 2010). A secretory tick ferritin, FER2, was sug-ested to be an iron transporter in the sheep tick Ixodes ricinusHajdusek et al., 2009) and in H. longicornis (Galay et al., 2013).

oreover, Hajdusek et al. (2009) and Galay et al. (2013) performedNA interference of ferritin-encoding genes, which resulted inbnormal reproduction. This supports the hypothesis that FER2ould be a main iron transporter to the ovaries in ticks. Galayt al. (2013) showed also that the intracellular H. longicornis fer-itin, HlFER1, which is implicated to be primarily responsible forntracellular iron storage, is expressed in various organs duringlood feeding, but not in the ovary of partially fed and fullyngorged adult females. Conversely, the secretory ferritin, HlFER2,hich is implicated to be responsible for iron transport, is weakly

xpressed during blood feeding, but increases during the ovipo-ition period in the ovary. Herein, we showed that host-derivedransferrin reached the ovary during blood feeding. Although theole of HlFER2 as an iron transporter is also still unclear, if it ishe case that host-derived transferrin transports iron to the ovary,e can hypothesize that host-derived transferrin synergistically

nteracts with HlFER2 in iron supplementation from host blood foritellogenesis.

The exact mechanism of the movement of rabbit-derived trans-errin into digestive cells and oocytes has not been thoroughlynvestigated, yet. It is believed that ticks have a number of routesf ingestion through the digestive cells by pinocytosis or by vari-us other types of endocytosis. However, little is known about theransport mechanism of host serum proteins (Sonenshine, 1991;effers and Michael Roe, 2008). Protein uptake by tick oocytes haseen studied in vitellogenin (Vg), which is synthesized mainly

n the fat body trophocytes and secreted into the hemolymph. Itas been believed that Vg is internalized by micropinocytosis andeposited into developing yolk protein in the oocytes without beingrocessed in the lysosomes (Coons et al., 1989). However, a specificg receptor was sequenced from the tick oocyte surface (Mitchellt al., 2007; Boldbaatar et al., 2008). In contrast, candidate homologsf the vertebrate transferrin receptors have not been detected inicks or insects, yet. Accordingly, H. longicornis may have a charac-eristic mechanism for the uptake of vertebrate transferrin into the

Please cite this article in press as: Mori, H., et al., Host-derived transferrin ilongicornis ticks. Ticks Tick-borne Dis. (2013), http://dx.doi.org/10.1016/j.t

ells that is different from that of vertebrates. Furthermore, the fatef rabbit-derived transferrin in the oocytes still remains unclearecause the laid eggs and the hatched larvae did not contain rabbit-erived transferrin. Provided that any transferrin receptor has not

PRESS Diseases xxx (2013) xxx– xxx 5

been identified, it could be considered that rabbit-derived trans-ferrin is processed within the lysosomes during the ovipositionperiod. More studies should be conducted to clarify the mecha-nisms of transport and/or the fate of host-derived proteins in tickbodies or tick organs, such as the midgut and ovary, which can leadto elucidation of definite functions of each protein.

In summary, we showed for the first time that rabbit-derivedtransferrin was persistently maintained in different developmen-tal stages and was transferred from the midgut to the hemolymphand the ovary of adult female H. longicornis. Additional experimentson mouse-fed adult female ticks further showed that host-derivedtransferrin can move from the midgut to the hemolymph andpass to the ovary. Although multiple functions of transferrin fromarthropods are suggested (Harizanova et al., 2005; Guz et al., 2007;Zhou et al., 2009), and most importantly, we have yet to know thefate of non-heme iron bound especially to host-derived transferrinin ticks, our present study showing the persistence of vertebratetransferrin in the tick raises the possibility that host-derived trans-ferrin is implicated in tick iron metabolism, particularly as aniron source and a transporter to the ovary, which may be cru-cial in vitellogenesis. Further investigation into the movement andmetabolism of host-derived proteins in ticks will help further elu-cidate tick survival strategies.

Supplementary data

Fig. S1. The specificity of the first antibody (sheep anti-rabbittransferrin IgG) was assured by Western blotting using sheep anti-rabbit transferrin IgG as the first antibody and HRP-conjugateddonkey anti-sheep IgG as the second antibody. Twelve �g of pro-tein extracts from engorged adult H. longicornis females and of totalprotein from blood (mouse and rabbit) and transferrin (bovine andrabbit) were loaded in each lane. Tick M, mouse-fed engorged adultfemale; Tick R, rabbit-fed engorged adult female; Blood M, mouseblood; Blood R, rabbit blood; Ba, bovine apo-transferrin; Bh, bovineholo-transferrin; Transferrin R, rabbit transferrin.

Fig. S2. Detection of rabbit-derived transferrin in thehemolymph (A) and in different organs (B) of partially fedand engorged adult female H. longicornis. The protein extracts(12 �g from organs, 16 �g from hemolymph) were loaded ineach lane, and then Western blot analysis was performed usingsheep anti-rabbit transferrin IgG as the first antibody and HRP-conjugated donkey anti-sheep IgG as the second antibody. Twelve�g of total protein from rabbit blood and from rabbit transferrinwere loaded as positive controls. Uf, unfed adult females; P1, 1-day-fed adult females; P2, 2-day-fed adult females; P3, 3-day-fedadult females; P4, 4-day-fed adult females; En1, adult females 1day after engorgement; RB, rabbit blood; RTF, rabbit transferrin.

Fig. S3. Detection of mouse-derived transferrin in whole bod-ies (A) and in the hemolymph and organs (the midgut and theovary) (B) of engorged adult female H. longicornis. Eight �g, 12 �gand 16 �g of protein extracts from whole bodies, organs and thehemolymph, respectively, were loaded in each lane, and thenWestern blot analysis was performed using rabbit-derived anti-mouse transferrin IgG as the first antibody and HRP-conjugateddonkey anti-rabbit IgG as the second antibody. Simultaneously,12 �g of total protein from rabbit and mouse blood, as well asapo-transferrin of bovine and mouse were loaded in each lane ascontrols. Arrowheads indicate positive bands of mouse transfer-rin. (C) Western blot analysis of the same tick samples using sheepanti-rabbit transferrin IgG as the first antibody and HRP-conjugateddonkey anti-sheep IgG as the second antibody. The numbers repre-

s maintained and transferred from midgut to ovary in Haemaphysalistbdis.2013.09.004

sent the days after engorgement. WB, whole body; HL, hemolymph;MG, midgut; OV, ovary; Uf, unfed adult females; Blood R, rabbitblood; Blood M, mouse blood; TF B, bovine apo-transferrin; TF M,mouse apo-transferrin.

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Fig. S4. Detection of rabbit-derived transferrin in eggs after lay-ng (A) and in larvae after hatching (B). The protein extracts (12g each) were loaded in each lane, and then Western blot analysisas performed using sheep anti-rabbit transferrin IgG as the first

ntibody and HRP-conjugated donkey anti-sheep IgG as the secondntibody. Twelve �g of total protein from rabbit blood and fromabbit transferrin were loaded as positive controls. (A) The num-ers represent the days after laying. (B) The numbers represent theays after hatching. RB, rabbit blood; RTF, rabbit transferrin.

cknowledgements

This study was supported by grants-in-aid for Scientificesearch (B) from the Japan Society for the Promotion of ScienceJSPS) and Morinaga Foundation.

ppendix A. Supplementary data

Supplementary material related to this article can be found, inhe online version, at doi:10.1016/j.ttbdis.2013.09.004.

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