M

IS

*

{

C H A P T E R T H I R T Y - F O U R

ethods

SN 0

LaboKagoDepa

Autophagy in Ticks

Rika Umemiya-Shirafuji,* Tomohide Matsuo,† and Kozo Fujisaki*

Contents

1. In

in

076

ratoshimrtme

troduction

Enzymology, Volume 451 # 2008

-6879, DOI: 10.1016/S0076-6879(08)03234-5 All rig

ry of Emerging Infectious Diseases, Department of Frontier Veterinary Medicine,a University, Kagoshima, Japannt of Infectious Diseases, Kyorin University School of Medicine, Tokyo, Japan

Else

hts

622

2. R

earing of the 3-Host Tick Haemaphysalis Longicornis 624

3. A

utophagy-Related Genes of H. longicornis 624

3

.1. C loning and identification of H. longicornis ATG genes 624

3

.2. E xpression and purification of recombinant HlAtg protein 626

3

.3. G eneration of anti-HlAtg12 antibody for immunoblotting and

immunofluorescence staining

627

4. E

xpression Profiles of HlATG12 from Nymphal to Adult Stages 627

4

.1. S emiquantitative reverse transcription-polymerase chain

reaction (RT-PCR)

627

5. D

etection of HlAtg Proteins in Midgut Cells 628

5

.1. Im munoblotting (Miyoshi et al., 2004; Umemiya et al., 2007b) 628

5

.2. Im munofluorescence staining for frozen sections

of the midgut

630

5

.3. Im muno-electron microscopy 631

6. U

ltrastructural Observation of Autophagosome- and Autolysosome-

like Structures in Midgut Cells of Unfed Ticks

633

7. C

onclusion 635

Ackn

owledgments 636

Refe

rences 636

Abstract

The generation time of ticks is estimated at several years and most ticks spend

more than 95% of their life off the host. They seem to have a unique strategy to

endure the off-host state for a long period. We focused on autophagy that is

induced by starvation and is essential for extension of the life span in model

organisms. Autophagy may occur in ticks that can survive extended periods of

starvation. Although little research has been done on autophagy in ticks,

recently, we showed the existence of an ATG gene homolog, HlATG12, in the

3-host tick Haemaphysalis longicornis. We have also examined the expression

vier Inc.

reserved.

621

622 Rika Umemiya-Shirafuji et al.

patterns of HlATG12, from nymphal to adult stages of this tick and revealed the

localization of the HlAtg12, protein within midgut epithelial cells of unfed adult

ticks. However, autophagy in ticks is a new field, so methods for monitoring this

phenomenon in ticks are still to be established. This chapter discusses proto-

cols for the detection of HlATG12, gene/HlAtg12, protein and the observation of

the midgut epithelial cells using an electron microscope during the nonfeeding

period of H. longicornis ticks. These methods can be adapted and modified for

the study autophagy in other hard ticks.

1. Introduction

Ticks are obligate hematophagous (blood feeding) arthropods foundin almost every region of the world (Sonenshine, 1991). They not onlyinfest every class of terrestrial vertebrates, including mammals, birds, variousreptiles and amphibians, but also are able to transmit various diseases to theseanimals. All ticks have 4 stages: the egg and three active stages (larva, nymphand adult). Each developmental stage except for the egg of most ticks feedson a host, and then drops off for development or reproduction in the naturalenvironment. Haemaphysalis longicornis is the most dominant tick in Japan,belongs to the hard tick family, and is categorized as a 3-host tick, whichfeeds on three different hosts in each stage. Engorged larvae drop off andmolt to nymphs, which then find a second host animal on which to engorgeand drop off again to molt into an adult. After new adults emerging fromengorged nymphs experience severe starvation, they attach to a third hostanimal (Fig. 34.1). Three-host ticks spend more than 95% of their life in thewild off the host, so their life cycle is characterized by a long period withoutfeeding, starvation (months or years), and an on-host parasitic period lastingonly a few weeks (Anderson, 2002). In particular, the period of host-seekingappears to be the longest in the adult stage of their life cycle.

Ticks are referred to as gorging-fasting organisms (Needham and Teel,1991), the only nourishment source for them is vertebrate blood. Unless bloodfeeding is fulfilled, ticks cannot develop and leave offspring for the nextgeneration. Once they take a blood meal, a variety of genes are up-regulatedand begin functioning. As a result, many investigators have studied fed ticks todevelop novel vaccines against ticks or tick-borne pathogens. However, theirastonishing toughness during the nonfeeding stage is extremely interesting.Details of the mechanisms by which ticks can survive such long periods areunknown, but it is suspected that ticks obtain nutrients by a uniquemechanismthat allows them to endure long-term starvation. Therefore, we have focusedon autophagy, which is induced by starvation, and hypothesize that ticks canadapt to long-term starvation by the mechanism of autophagy (autophagymeaning macroautophagy in this chapter).

Unfed nymph(several months)

Unfed adult(several monthsor years)Feeding

(several days) (2 weeks)

Molting(2 weeks)

Engorgedlarva

Unfed larva(several months)

Engorgedadult

Feeding(1 week)

Feeding(several days)

Egg

Hatching

Oviposition

Death

MoltingEngorgednymph

Figure 34.1 Life cycle of a 3-host tick,H. longicornis.The life cycle consists of 4 stages,the egg and three active stages: larva, nymph and adult. Larvae and nymphs feed oncefor several days to engorgement and then molt whereas adults feed for approximately1week.The engorged larvae drop off, molt to a nymph stage and find a second host onwhich to engorge and drop off again tomolt to an adult.The adult female ticks attach toand engorge on a third host, and then finally die after oviposition, which occurs off thehost. Note that most of their life cycle is characterized by a long nonfeeding period,starvation.

Autophagy in Ticks 623

Autophagy in ticks has not been reported, but genes encoding ubiquitinhave been reported in the transcriptome analysis of tick salivary glands(Alarcon-Chaidez et al., 2007; Francischetti et al., 2005). The presence ofautophagic vacuoles in the midgut, the digestive organ of ticks, has beendetected by electron microscopy (Tarnowski and Coons, 1989; Walker andFletcher, 1987), but only the fine structure of the vacuoles was determined.The midgut cells of unfed ticks have three types of autophagic vacuoles;type 1 with spheroid inclusions, type 2 with lamellate inclusions, and type 3with excretory inclusions (Raikhel, 1983). These structures seem to appearonly during the unfed stages. However, questions about their function(s)and how they are related to autophagy remain unanswered.

624 Rika Umemiya-Shirafuji et al.

We first isolated homologs of autophagy-related (ATG) genes and thencloned a homologue of an ATG gene (ATG12), designated as HlATG12,from the hard tickH. longicornis (Ummeiya et al., 2007b), a vector of Babesiaand Theileria parasites, and rickettsia. In this chapter, we describe methodsfor the detection of an autophagy-related gene/protein (HlATG12/HlAtg12) and the observation of autophagosome- or autolysosome-likestructures in the midgut epithelial cells of the 3-host tick, H. longicornis.

2. Rearing of the 3-Host Tick HaemaphysalisLongicornis

Adult parthenogenetic H. longicornis ticks (Okayama strain) were main-tained by feeding on the ears of Japanese white rabbits (3.0 kg, female; JapanLaboratory Animals, Tokyo, Japan) protected with cotton bags. Engorgedticks that dropped off the animals were collected and maintained toallow them to molt or lay eggs. Tick-rearing was performed in an incubatorat 25 �C, 100% relative humidity and continuous darkness (Fujisaki, 1978).

3. Autophagy-Related Genes of H. longicornis

3.1. Cloning and identification of H. longicornis ATG genes

Although tick research has also entered the genomic era, genome sequenc-ing of only a few tick species is in progress because of unexpectedly largetick genomes ( Jongejan et al., 2007) similar in size or several times largerthan the human genome. For tick sequence analysis, the EST gene indicesare the best sources for identifying potential homologs at present becausesequencing of the genome is still far from completion. Therefore, theconstruction of cDNA libraries and the generation of EST databases forH. longicornis were obtained from Hitachi High-Tech Manufacturing &Service (Ibaraki, Japan) using proprietary protocols. We isolated 4 ATGgene homologs for H. longicornis from these databases (Umemiya et al.,2007b; Umemiya-Shirafuji et al., unpublished results). The protocol toconstruct a cDNA library by the vector-capping method (Kato et al.,2005) is simply outlined subsequently.

1. Extract total RNAs (5 mg) from each organ of partially fed adult femalesusing a TRI reagent (Sigma, MO, USA) as follows:a. Homogenize tissue samples with a mortar and pestle (AS ONE,

Osaka, Japan) using liquid nitrogen and resuspend in TRI reagent(1 ml per 50–100 mg of tissue).

Autophagy in Ticks 625

b. Centrifuge the extracts for 10 min, 4 �C at top speed. Transferaqueous phase to a new tube.

c. Add chloroform (0.2 ml per 1 ml of TRI reagent) to the suspension.Shake for 15 s and allow to stand for 15 min at room temperature.Centrifuge the resulting mixture at top speed for 15 min at 4 �C.

d. Transfer the aqueous phase to a new tube and add isopropanol (0.5 mlper 1 ml of TRI reagent) and mix. Allow the sample to stand for10 min at room temperature. Centrifuge at top speed for 10 min at4 �C.

e. Remove the supernatant fraction and wash the RNA pellet by adding75% ethanol (1 ml per 1 ml of TRI reagent). Vortex the sample andthen centrifuge for 5 min, 4 �C at top speed.

f. Remove the supernatant fraction and dry the RNA pellet for 5 minby air-drying.

g. Add an appropriate volume of nuclease-free water to the pellet.2. Synthesize cDNAs and ligate into the pGCAP1 plasmid vector (Hitachi

High-Tech Manufacturing & Service).3. Transform into Escherichia coli (DH12S strain).4. Select bacterial colonies and inoculate into 384 well plates containing

approximately 0.1 ml of LB medium.5. Isolate and sequence plasmid DNAs.6. Perform annotation by searching public protein databases using the

BLAST analysis (National Center for Biotechnology Information;NCBI) to identify a provisional function. (See Kato et al., 2005, forexperimental details, especially for the procedure from steps 2–6.

Finally, the EST databases of H. longicornis were constructed. The Micro-soft Office Excel software was adopted for using the databases, which areavailable at the Laboratory of Emerging Infectious Diseases, KagoshimaUniversity. The databases have not been accessible but will be available onlinein the future. To isolate pure plasmids from E. coli, a miniprep kit such as aQIAprep Spin Miniprep Kit (Qiagen, Hilden, Germany) is useful. Thefollowing tools are helpful for characterization of cDNAs: GENETYX ver-sion 7 software (Genetyx, Tokyo, Japan), BLAST analysis (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi; NCBI), ClustalW2 (http://www.ebi.ac.uk/Tools/clustalw2/index.html; European Bioinformatics Institute (EMBL-EBI)), ExPASy Proteomics Server (http://kr.expasy.org/; Swiss Institute ofBioinformatics (SIB)), and SignalP 3.0 (http://www.cbs.dtu.dk/services/SignalP/; Center for Biological Sequence Analysis (CBS)).

In addition, some novel genes have been previously isolated fromH. longicornis by using the databases (Alim et al., 2007, 2008; Boldbaataret al., 2006; Gong et al., 2007; Harnnoi et al., 2007; Liao et al., 2007a,b;Motobu et al., 2007; Tanaka et al., 2007; Umemiya et al., 2007a,b; Zhou et al.,2006, 2007). The identification of numerous genes from the databases will

626 Rika Umemiya-Shirafuji et al.

help in the elucidation of the mystery by which ticks survive for long periodswithout feeding.

3.2. Expression and purification of recombinant HlAtg protein

The protocol for expression of a recombinant HlAtg protein, specificallyHlAtg12 (Umemiya et al., 2007b), for generation of antibodies is describedsubsequently.

1. DNA for subcloning into the vector plasmid pGEX-4T-3 (GE Health-care, Chalfont St. Giles, UK) is prepared by conventional methods,utilizing the polymerase chain reaction (PCR) and digestion with restric-tion enzymes. After subcloning, recombinant plasmids should be con-firmed by sequence analysis.

2. The plasmid is then transformed into E. coli (BL21 strain) to express therecombinant protein from pGEX-4T-3. The transformants are selectedby the antibiotic ampicillin.

3. Synthesis of recombinant glutathione S-transferase (GST)-fused HlAtg12(GST-HlAtg12) is induced with 0.05 mM isopropyl-b-D-thiogalacto-pyranoside for 4 h at 37 �C.

4. After centrifugation at 8000g for 20 min at 4 �C, 20–30 ml of TBST(50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100) contain-ing 2 mM dithiothreitol and a proteinase inhibitor (Complete Mini;Roche Diagnostics, Mannheim, Germany) per 1 liter of culture isadded to the bacterial pellet.

5. The suspension is sonicated on ice using a sonicator (10 s � 3), andcentrifuged at 10,000g for 10–30 min at 4 �C.

6. The supernatant fraction is transferred to a new tube and filtered throughsterile 5- and 0.22-micron filters (Toyo Roshi, Tokyo, Japan).

7. Soluble GST-HlAtg12 is obtained using batch purification as follows:a. 0.5–1 ml of the 50% slurry of Glutathione Sepharose

Ò4B (GE Health-

care) equilibrated with TBST per 1 liter of culture is added to thecleared lysate and mixed gently using a rotator at 4 �C, overnight.

b. The mixture is centrifuged at 500g for 5 min at 4 �C.c. After removal of the supernatant fraction, the sedimented Glutathione

SepharoseÒ4B is washed with 10 bed volumes of TBST 3 times.

d. The supernatant fraction is discard and then 16 mM reduced gluta-thione in 50 mM Tris-HCl, pH 8.0 (1 ml per bed volume of Gluta-thione Sepharose 4B) is added to the sedimented matrix.

e. The mixture is incubated at room temperature for 10 min, thencentrifuged at 500g for 5 min.

f. The supernatant fraction containing GST-HlAtg12 is transferred to anew tube.

Autophagy in Ticks 627

3.3. Generation of anti-HlAtg12 antibody for immunoblottingand immunofluorescence staining

Purified GST-HlAtg12 is dialyzed against Tris-buffered saline adjusted topH 7.4. The protein concentration is determined by a bicinchoninic acidassay (Pierce Biotechnology, IL, USA). One hundred micrograms of theGST-HlAtg12 in Freund’s complete adjuvant (Sigma-Aldrich, MO, USA)are used to immunize mice (ddY, 6 weeks old, female; Japan LaboratoryAnimals). The same antigen in Freund’s incomplete adjuvant (Sigma-Aldrich)is repeatedly injected intraperitoneally into the mice on days 14 and 28. Seraare collected from these mice 10 days after the last immunization. Theantibody titer can be confirmed by an enzyme-linked immunosorbent assayor Western blot analysis.

4. Expression Profiles of HlATG12 from

Nymphal to Adult Stages

4.1. Semiquantitative reverse transcription-polymerase chainreaction (RT-PCR)

4.1.1. RNA extraction and cDNA synthesisFigure 34.2 shows the outline of sampling.

1. Nymphal ticks infesting on rabbits are detached and used on days 1, 2,and 3 after attachment. In addition, engorged nymphs are collected andthen a part of them are maintained in a moist chamber at 25 �C. Somemaintained ticks are collected at 10 days (premolting), and the others are

Blood feeding

Unfednymph

0d 1d 2d 3d 10d 20d

Period after engorgement of nymph

1mo 3mo

Unfedadult

Engorgednymph

Molting

Figure 34.2 Outline for sampling of nymphal and adult stages. Black arrows indicatethe timing of sample collection of ticks.Ticks were collected unfed, 1-, 2-, and 3-days-fed, and 10 days (premolting) after engorgement and the others were allowed to moltunder the same conditions. Molted new emerging adult ticks were collected at 20 days(postmolting), 1 and 3 months after engorgement in the nymphal stage. d, day(s); mo,month(s).

628 Rika Umemiya-Shirafuji et al.

allowed to molt in a moist chamber at 25 �C. Molted ticks are collected20 days, 1 month, and 3 months after engorgement.

2. Ten ticks collected in each stage are homogenized with a mortar andpestle (AS ONE, Osaka, Japan) using liquid nitrogen and are resus-pended in TRI reagent (SIGMA) as described earlier.

3. Total RNA is extracted from the suspensions, and in order to removegenomic DNA, the sample is treated with DNase (TURBO DNA-freeKit; Applied Biosystems, CA, USA). Briefly, DNase (2 units for up to10 mg of RNA) are added to the RNA solution and mixed gently, thenincubated at 37 �C for 20–30 min.

4. Single-strand cDNA is generated from the treated total RNA (10 ng–5 mg) by reverse transcription using Transcriptor First Strand cDNASynthesis Kit (Roche Diagnostics, Mannheim, Germany). This kit con-tains oligo(dT)18 primer, RNase inhibitor, deoxynucleotide mix, andtrascriptor reverse transcriptase which is able to synthesize long cDNAproducts (up to 14 kDa). The reaction proceeds as follows: 65 �C for10 min, 50 �C for 60 min, and 85 �C for 5 min.

4.1.2. Polymerase chain reaction (PCR) for amplification of HlATG12PCR is carried out with the appropriate dilutions of synthesized cDNAs andHlATG12-specific primers (sense primer, 50-ATGTCCGATGAAACTGAAGGCTGTGCGACTGCG-30; antisense primer, 50-TTAGCCCCATGCGTGACTTTTTGCATAGTGCAGAG-30). PCR conditions are94 �C for 30 s, 60 �C for 30 s, and 72 �C for 1 min for 30 cycles. Controlamplification is carried out using the specific primers (sense primer, 50-CCAACAGGGAGAAGATGACG-30; antisense primer, 50-ACAGGTCCTTACGGATGTCC-30) designed from H. longicornis actin (Da Silva Vaz Jr.et al., 2005). The PCR products are electrophoresed on a 1.5% agarose gel andstained with ethidium bromide, and then the gel image is digitized for densi-tometry analysis by Luminous Imager Software (version 2.0; Aisin CosmosR&D, Aichi, Japan). The results are expressed as a ratio of the density of theHlATG12 products to the density of the actin products from the same template.

5. Detection of HlAtg Proteins in Midgut Cells

5.1. Immunoblotting (Miyoshi et al., 2004; Umemiya et al.,2007b)

5.1.1. Extraction of soluble proteins from whole ticks

1. Homogenize 5 unfed adult ticks with a mortar and pestle (AS ONE) inliquid nitrogen and resuspend in 0.1 ml of phosphate-buffered saline

Autophagy in Ticks 629

(PBS, pH7.4) containing a proteinase inhibitor (Complete Mini; RocheDiagnostics, Mannheim, Germany).

2. Transfer the lysate to a centrifuge tube and sonicate for 30 s using awater-bath sonicator.

3. Centrifuge the extracts at 26,000g for 30 min using a high-speed micro-centrifuge (Hitachi Koki, Tokyo, Japan). Store supernatants at �30 �Cuntil used for immunoblotting.

5.1.2. Cell fractionation of the midgut by differential centrifugationThe midgut of ticks demonstrates multifunctional activity. Digestion of hostblood begins during the slow feeding period (a few days after attachment)and digestion is accomplished slowly within epithelial cells of the midgut(Sonenshine, 1991). However, the protein content in ticks graduallydecreases until the tick encounters the next suitable host. In addition tothe main digestive function, the midgut serves as the major deposit ofnutritional reserves represented by intracellular inclusions of host bloodhemoglobin, lipids and carbohydrates. In female ticks, the yolk proteinsare synthesized in the midgut as well as the fat body. Unlike insects, manycorresponding metabolic processes probably occur in the midgut cells ofticks (Raikhel, 1983). Moreover, the midgut is the first organ that patho-gens invade and develop within. There are various genes that showenhanced expression in response to pathogens in the midgut, for examplethe tick receptor for OspA (TROSPA) within the midgut of Ixodes scapularis(Pal et al., 2004). Consequently, the midgut is a very important organ forexamining autophagy in ticks. The protocol to examine the distribution ofHlAtg12 protein within the midgut cells is explained subsequently.

1. Dissect 10 unfed adult ticks in 3–5 ml of PBS containing a proteinaseinhibitor (Complete Mini; Roche Diagnostics).

2. Homogenize the midguts using a glass homogenizer (AS ONE) in 0.1 mlof PBS containing a proteinase inhibitor with 0.25 M sucrose on ice.

3. Centrifuge at 1000g for 7 min at 4 �C to generate a pellet (P1; thefraction-containing the nucleus and unhomogenized cells).

4. Centrifuge the resulting supernatant fraction at 2000g for 30 min at 4 �Cto generate a pellet (P2; the fraction containing the mitochondria andlysosomes).

5. Transfer the supernatant fraction to a special tube for the ultracentrifugeand centrifuge at 105,000g for 60 min at 4 �C to generate a pellet (P105;the fraction containing membranes and ribosomes) using a Micro Ultra-centrifuge (Hitachi Koki, Tokyo, Japan).

6. Transfer the supernatant fraction (S105; the fraction containing cytosol)to a new tube. Store samples at �30 �C until used for immunoblotting.

630 Rika Umemiya-Shirafuji et al.

5.1.3. SDS-PAGE and Immunoblotting for detection of HlAtg12The tick proteins extracted by the preceding methods are separated by SDS-PAGE under nonreducing and/or reducing (in the presence of2-mercaptoethanol) conditions on a 5%–20% gradient acrylamide gel(e�PAGELÒ; ATTO, Tokyo, Japan) and analyzed by Western blot.

1. Separated proteins are transferred from gels onto a PVDF membrane bysemidry blotting.

2. The membrane is incubated with 3% (w/v) skim milk in PBS for 1 h atroom temperature.

3. After washing with PBS containing 0.1% (v/v) Tween 20 (PBST), themembrane is incubated with a suitable dilution of mouse polyclonal anti-GST-HlAtg12 antibody (Umemiya et al., 2007b) for 1 h at roomtemperature.

4. The membrane is thoroughly washed with >4 ml/cm2 of PBST 3 timesand the binding of antibody is detected with horseradish peroxidase-conjugated polyclonal goat antimouse immunoglobulin (Dako,Glostrup, Denmark) at a dilution of 1:10000 and an ECL AdvanceWestern Blotting Detection Kit (GE Healthcare). Preliminary measure-ments must be taken to determine the proper dilution because the ECLadvance kit is highly sensitive.

5. The images are analyzed using VersaDoc Model 500 (Bio-Rad Labora-tories, Tokyo, Japan).

5.2. Immunofluorescence staining for frozen sectionsof the midgut

1. Dissect and excise the midgut from unfed and 4-day-fed adult femaleticks.

2. Fix with fixative (4% [w/v] paraformaldehyde including 0.1% glutaral-dehyde in PBS) in each 10-ml sample bottle at 4 �C, overnight byshaking gently.

3. Wash the midguts with PBS (5 min � 3).4. Soak in 5%, 10%, 15%, and 20% sucrose in PBS at 4 �C.

Note: The samples should be soaked in each concentration of sucrose

solution until they sink as much as possible (more than 4 h).

5. Make a containermatching the size of themidgut with aluminum foil, andpour chilled Tissue-Tek O.C.T. Compound (Sakura Finetek Japan,Tokyo, Japan) into the container up to about 80%. Submerge the fixedmidgut in the compound. Embed each midgut in Tissue-Tek O.C.T.Compound with liquid nitrogen. Keep frozen blocks at �80 �C.

Note: The samples should be submerged in Tissue-Tek O.C.T.

Compound for at least 30 min before freezing.

Autophagy in Ticks 631

6. Cut frozen sections (approximately 14 mm thick) on a cryostat (Leica CM3050; Leica Microsystems, Wetzlar, Germany) and then place them onaminosilanes-coated glass slides (Matsunami Glass, Osaka, Japan).

7. Dry for 30 min or more by air-drying.8. Wash with PBS (5 min � 3) in a jar. The amount of PBS depends on

the size of the jar used.9. Block with 3%–5% (w/v) skimmilk in PBS at room temperature for 1 h

on the glass slide or in a jar.10. Immunostain

a. Incubate with a suitable dilution of primary antibody (mouse anti-GST-HlAtg12 antibody) overnight at room temperature for 1 h orat 4 �C. Because the dilution factor depends on the antibody titer,preliminary testing is needed.

b. Wash with PBS (5 min � 3) in a jar.c. Incubate with Alexa Fluor 594 conjugated goat anti-mouse IgG

(1:1000; Molecular Probes, OR, USA) at room temperature for1–2 h.

d. Wash with PBS (5 min � 3).11. Observe with a confocal laser-scanning microscope (TCS NT, Leica

Microsystems) or an immunofluorescence microscope.

The anti-GST-HlAtg12 antibody reacts with the cytoplasm of themidgut epithelial cells, which seem to be digestive cells, during immuno-fluorescence staining. Moreover, positive reactions appear as some aggrega-tions of red-colored dots in digestive cells of unfed adults. In contrast, thedots are not observed in the digestive cells of 4-day-fed adults even thoughthe anti-GST-HlAtg12 antibody slightly reacts with the cytoplasm. Positivedots observed in unfed samples are very small (less than 1 mm in diameter),so we recommend the observation of the midgut cells by electron micros-copy as described in the following methods.

5.3. Immuno-electron microscopy

5.3.1. Preparation of samples from unfed ticks

1. Dissect unfed female ticks as described previously.2. Fix small pieces of midguts with 4% paraformaldehyde containing 0.1%

glutaraldehyde in PBS (pH 7.4) in each 10-ml sample bottle at 4 �C,overnight, by shaking gently.

Note: It is difficult for the fixative to infiltrate into the tick midgut. To

deal with this problem, if possible, air should be removed from the midgutwith a vacuum pump or an evaporator. Such treatment for more than30 min will ensure the fixation buffer reaches inside the lumen. Thismethod is also effective for the infiltration of resin during embedding.

632 Rika Umemiya-Shirafuji et al.

3. Remove the fixative and wash with PBS in the bottle at 4 �C by shakinggently (�30 min � 3).

4. Remove the PBS and dehydrate with an ethanol series shown below inthe bottle.a. 30% ethanol for �10 min at 4 �Cb. 50% ethanol for �10 min at 4 �Cc. 70% ethanol for �10 min at 4 �Cd. 80% ethanol for �10 min at 4 �Ce. 90% ethanol for �10 min at �20 �Cf. 99% ethanol for �10 min at �20 �Cg. 100% ethanol for �20 min � 3 at �20 �C

5. Remove the 100% ethanol and embed in LR gold resin (Polysciences,PA, USA) as indicated subsequently.a. The mixture of 100% ethanol and LR gold resin (1:2) at �20 �C,

overnightb. The mixture of 100% ethanol and LR gold resin (1:1) at �20 �C,

overnightc. The mixture of 100% ethanol and LR gold resin (2:1) at �20 �C,

overnightd. Transfer the midgut sample to a gelatin capsule (Nisshin EM, Tokyo,

Japan) where 1 drop of pure LR gold resin was placed with a 1-mlsyringe.

e. Fill the capsule with LR gold resin and incubate at �20 �C for 2–3days (until polymerization of the resin is completed) under UV light.

6. Cut ultrathin sections (approximately 70-nm thick).7. Place the sections on nickel grids (Nisshin EM).

5.3.2. Immunostain

1. Block with 5% (w/v) skim milk in PBS.2. Incubate the sections overnight by floating the grids on a 100-ml drop of

a suitable dilution of primary antibody (anti-GST-HlAtg12 antibody)at 4 �C.

3. Wash three times with 100 ml of PBS.4. Incubate the grids in a 100-ml drop of 10-nm gold-conjugated goat

antimouse IgG + IgM (1:30; GE Healthcare) at room temperaturefor 2 h.

5. Wash three times with 100 ml of PBS.6. Fix with a 100-ml drop of 3% (w/v) glutaraldehyde in PBS for 15 min.7. Wash three times with 100 ml of distilled water.8. Stain with a 100-ml drop of 5% uranyl acetate in 50% ethanol for 5 min.9. Observe the sections with a JEM-1010 electron microscope ( JEOL,

Tokyo, Japan).

Autophagy in Ticks 633

By using the preceding methods, gold particles indicate the positive sitesshowing cytoplasmic distribution, and the anti-GST-HlAtg12 antibodyreacts with the exterior of the granulelike structures. These structures aresmall and comparatively electron dense. The size of most granulelikestructures is approximately 500 nm or less (Umemiya et al., 2007b).

6. Ultrastructural Observation of

Autophagosome- and Autolysosome-like

Structures in Midgut Cells of Unfed Ticks

It is essential to observe the autophagic structures with an electronmicroscope to be able to describe autophagy in ticks.

1. Dissect unfed female ticks as described previously.2. Fix small pieces of midguts overnight with 3% glutaraldehyde in 0.1 M

cacodylate buffer (pH 7.4) in each 10-ml sample bottle at 4 �C byshaking gently.

Note: If possible, air should be removed from the midgut with a vacuum

or an evaporator as described earlier.

3. Remove the fixative and wash with 0.1 M cacodylate buffer in the bottleby shaking gently (�30 min � 3).

4. Postfix with approximately 3 ml of 1% OsO4 in the same buffer for 2 h at4 �C.

5. Dehydrate with an ethanol series as here.a. 30% ethanol for �10 min at 4 �Cb. 50% ethanol for �10 min at 4 �Cc. 70% ethanol for �10 min at 4 �Cd. 80% ethanol for �10 min at 4 �Ce. 90% ethanol for �10 min at room temperaturef. 99% ethanol for �10 min at room temperatureg. 100% ethanol for �20 min � 3 at room temperatureh. 100% propylene oxide

6. Remove the 100% propylene oxide and embed in Epon resin (NisshinEM) as follows:a. The mixture of 100% propylene oxide and Epon resin (1:2) for half a

day at room temperatureb. The mixture of 100% propylene oxide and Epon resin resin (1:1) for

half a day at room temperaturec. The mixture of 100% propylene oxide and Epon resin resin (2:1) for

half a day at room temperatured. Transfer the midgut sample to the BEAM capsule (Nisshin EM)

where 1 drop of pure Epon resin was placed with a 1-ml syringe.

634 Rika Umemiya-Shirafuji et al.

e. Fill the capsule with Epon resin and incubate at 60 �C for 2 days (untilpolymerization of the resin is completed).

7. Cut ultrathin sections (approximately 80 nm thick) and place on coppergrids (Nisshin EM).

8. Double stain with 100 ml of 5% uranyl acetate in 50% ethanol and leadcitrate (Reynold’s method) for 5 min.

9. Observe the sections with an electron microscope ( JEM-1010; JEOL).

The preceding techniques reveal some types of autophagic organelles indigestive cells of unfed females (Figs. 34.3 and 34.4). Most of these struc-tures are approximately 1–2 mm or smaller, surrounded by a single mem-brane (Fig. 34.3A, arrows), and include almost normal cytoplasm with smalldense granules. This appearance seems like an amphisome rather than anautophagosome. Amphisomes are also autophagic organelles surrounded bya single membrane that are formed by the fusion of autophagosomes andearly or late endosomes (Berg et al., 1998; Klionsky et al., 2007). Compari-son with previous observations of tick midgut cells indicates the structureshown in Fig. 34.3B is a type 1 autophagic compartment (arrowheads). Thetype 1 inclusions (0.5-2.0 mm in diameter) have a fine granular material oflow density and a dense rim under the surrounding membrane (Raikhel,1983). On the other hand, the structures containing a layerlike form(Fig. 34.3B, arrows) appears to be consistent with myelinosiderome,

G

G

0.5mm 1mm

A B

Figure 34.3 Autophagic vacuoles in the midgut epithelial cells of unfed female adultticks. (A) The structure is surrounded by a single membrane (arrows). The content isalmost normal cytoplasm. (B) Two kinds of autophagic vacuoles, one a lucent vacuole(arrowheads), and the other a structure that contains layers of various densities (arrows).Note the double membrane surrounding the lucent structures. Single arrowheads anddouble arrowheads indicate the outer and innermembranes, respectively. G, granules.

G

G

1mm0.5mm

A B

Figure 34.4 Autolysosome-like structures containing recognizable remnants of cyto-plasmic elements in the midgut epithelial cells of unfed female adult ticks. (A) Thestructure is surrounded by a single membrane (arrows) and contains relatively largeelectron-dense granules. (B) Arrow indicates a single membrane of the autolysosome-like structure.The cytoplasmic contents appear to be denatured. G, granules.

Autophagy in Ticks 635

which are residual bodies containing myelin figures and hemosiderin fromhemoglobin degradation (Williams et al., 1985). Additionally, theautolysosome-like structures were found in the cytoplasm of the midgut,which is surrounded by a single membrane and contains relatively largeelectron-dense granules (Fig. 34.4). Although these unique structures areoften found inmidgut cells of unfed ticks, their function(s) are still unknown.Comparative observations on unfed midgut cells by both electron andimmunoelectron microscopy will lead to better understanding as to whetherthere is a relationship between the HlAtg proteins and autophagic vesicles.

7. Conclusion

The protocols described in this chapter can be used for investigation ofautophagy in ticks. Observations using electron microscopy are the mostsuitable method for detection of autophagic organelles in the midgut ofticks. No standard methods are established for ticks because the study ofautophagy in ticks has just begun. There are many useful and convenientmethods that can be used to monitor macroautophagy in yeast, but rela-tively few in other model systems, and there is much confusion regardingacceptable methods to measure macroautophagy in higher eukaryotes

636 Rika Umemiya-Shirafuji et al.

(Klionsky et al., 2008). In addition, autophagy is a dynamic, multistepprocess that can be both positively and negatively modulated at severalsteps. If midgut cells can be cultured in vitro, the autophagic processes maybe monitored in more detail. Phagophores (isolation membranes) are easilyrecognized as ultrastructurally compressed (electron dense), curving cister-nae during the process of enclosing of the cytoplasm in mammalian cells(Klionsky et al., 2007). Unfortunately, the phagophore-like structure hasnot yet been identified in tick midgut cells.

Autophagy is rapidly induced by starvation in the larval fat body (Scottet al., 2004), which is known to be the nutrient storage organ. Whilelysosomes in Drosophila fat body cells are small and few in number underfed conditions, the lysosomes increase and enlarge, and autolysosomesrapidly form during starvation. Lysosomal staining is useful in Drosophila asa proxy method for monitoring autophagy but this method is not alwayspossible in ticks because ticks have an intracellular digestive system fordegradation of host blood. The digestion via lysosomes takes place activelyin the cytoplasm of midgut cells during and after a blood meal because themidgut is the principle digestive organ (Mendiola et al., 1996). Therefore, itis not always possible to use changes in lysosomes in order to monitorautophagy in ticks.

We have identified homologs of ATG3, ATG4 andATG8 genes as wellas ATG12 from ESTs constructed from a cDNA library of H. longicornis(Umemiya et al., 2007b; Umemiya-Shirafuji et al., unpublished results).Particularly, further investigation of the ATG8 homolog, which is a markerfor autophagosomes, will provide additional insight into the roles of thesemolecules in the blood-feeding physiology of ticks (Umemiya et al., 2008),but further trials are required to establish the appropriate methods tomonitor autophagy in ticks.

ACKNOWLEDGMENTS

This study was supported by the Bio-oriented Technology Research Advancement Institu-tion (BRAIN), a Grant-in-Aid for Scientific Research (A) from the Japan Society for thePromotion of Science, and a grant from the 21st Century COE program (A-1), the Ministryof Education, Sports, Science, and Technology of Japan. The first author was supported by aGrant-in-Aid for JSPS Fellows from the Japan Society for the Promotion of Science ( JSPS).We also thank Dr. DeMar Taylor (University of Tsukuba) for special advice with regard tomanuscript improvement.

REFERENCES

Alarcon-Chaidez, F. J., Sun, J., and Wikel, S. K. (2007). Transcriptome analysis of thesalivary glands of Dermacentor andersoni Stiles (Acari: Ixodidae). Insect Biochem. Mol. Biol.37, 48–71.

Autophagy in Ticks 637

Alim, M. A., Tsuji, N., Miyoshi, T., Islam, M. K., Huang, X., Motobu, M., and Fujisaki, K.(2007). Characterization of asparaginyl endopeptidase, legumain induced by blood feed-ing in the ixodid tick Haemaphysalis longicornis. Insect Biochem. Mol. Biol. 37, 911–922.

Alim, M. A., Tsuji, N., Miyoshi, T., Islam, M. K., Huang, X., Hatta, T., and Fujisaki, K.(2008). HlLgm2, a member of asparaginyl endopeptidases/legumains in the midgut of theixodid tick Haemaphysalis longicornis, is involved in blood-meal digestion. J. Insect Physiol.54, 573–585.

Anderson, J. F. (2002). The natural history of ticks. Med. Clin. North Am. 86, 205–218.Berg, T. O., Fengsrud, M., Str�mhaug, P. E., Berg, T., and Seglen, P. O. (1998). Isolation

and characterization of rat liver amphisomes: Evidence for fusion of autophagosomeswith both early and late endosomes. J. Biol. Chem. 273, 21883–21892.

Boldbaatar, D., Sikasunge, C. S., Battsetseg, B., Xuan, X., and Fujisaki, K. (2006). Molecularcloning and functional characterization of an aspartic protease from the hard tick Hae-maphysalis longicornis. Insect Biochem. Mol. Biol. 36, 25–36.

Da Silva Vaz, I., Imamura, S., Nakajima, C., De Cardoso, F. C., Ferreira, C. A., Renard, G.,Masuda, A., Ohashi, K., and Onuma, M. (2005). Molecular cloning and sequenceanalysis of cDNAs encoding for Boophilus microplus, Haemaphysalis longicornis and Rhipi-cephalus appendiculatus actins. Vet. Parasitol. 127, 147–155.

Francischetti, I. M., My-Pham, V., Mans, B. J., Andersen, J. F., Mather, T. N., Lane, R. S.,and Ribeiro, J. M. (2005). The transcriptome of the salivary glands of the female westernblack-legged tick Ixodes pacificus (Acari: Ixodidae). Insect Biochem. Mol. Biol. 35,1142–1161.

Fujisaki, K. (1978). Development of acquired resistance and precipilating antibody in rabbitsexperimentally infested with females of Haemaphysalis longicornis (Ixodoidea: Ixodidae).Natl. Inst. Anim. Health Q (Tokyo) 18, 27–38.

Gong, H., Zhou, J., Liao, M., Hatta, T., Harnnoi, T., Umemiya, R., Inoue, N., Xuan, X.,and Fujisaki, K. (2007). Characterization of a carboxypeptidase inhibitor from the tickHaemaphysalis longicornis. J. Insect Physiol. 53, 1079–1087.

Harnnoi, T., Sakaguchi, T., Nishikawa, Y., Xuan, X., and Fujisaki, K. (2007). Molecularcharacterization and comparative study of 6 salivary gland metalloproteases from the hardtick, Haemaphysalis longicornis. Comp. Biochem. Physiol. B, Biochem. Mol. Biol. 147,93–101.

Jongejan, F., Nene, V., de la Fuente, J., Pain, A., and Willadsen, P. (2007). Advances in thegenomics of ticks and tick-borne pathogens. Trends Parasitol. 23, 391–396.

Kato, S., Ohtoko, K., Ohtake, H., and Kimura, T. (2005). Vector-capping: a simple methodfor preparing a high-quality full-length cDNA library. DNA Res. 12, 53–62.

Klionsky, D. J., Cuervo, A. M., and Seglen, P. O. (2007). Methods for monitoringautophagy from yeast to human. Autophagy 3, 181–206.

Klionsky, D. J., Abeliovich, H., Agostinis, P., Agrawal, D. K., Aliev, G., Askew, D. S.,Baba, M., Baehrecke, E. H., Bahr, B. A., Ballabio, A., Bamber, B. A., Bassham, D. C.,et al. (2008). Guidelines for the use and interpretation of assays for monitoring autophagyin higher eukaryotes. Autophagy 4, 151–175.

Liao, M., Zhou, J., Hatta, T., Umemiya, R., Miyoshi, T., Tsuji, N., Xuan, X., andFujisaki, K. (2007a). Molecular characterization of Rhipicephalus (Boophilus) microplusBm86 homologue from Haemaphysalis longicornis ticks. Vet. Parasitol. 146, 148–157.

Liao, M., Hatta, T., Umemiya, R., Huang, P., Jia, H., Gong, H., Zhou, J., Nishikawa, Y.,Xuan, X., and Fujisaki, K. (2007b). Identification of three protein disulfide isomerasemembers from Haemaphysalis longicornis tick. Insect Biochem. Mol. Biol. 37, 641–654.

Mendiola, J., Alonso, M., Marquetti, M. C., and Finlay, C. (1996). Boophilus microplus:Multiple proteolytic activities in the midgut. Exp. Parasitol. 82, 27–33.

638 Rika Umemiya-Shirafuji et al.

Miyoshi, T., Tsuji, N., Islam, M. K., Kamio, T., and Fujisaki, K. (2004). Cloning andmolecular characterization of a cubilin-related serine proteinase from the hard tickHaemaphysalis longicornis. Insect Biochem. Mol. Biol. 34, 799–808.

Motobu, M., Tsuji, N., Miyoshi, T., Huang, X., Islam, M. K., Alim, M. A., and Fujisaki, K.(2007). Molecular characterization of a blood-induced serine carboxypeptidase from theixodid tick Haemaphysalis longicornis. FEBS Lett. 274, 3299–3312.

Needham, G. R., and Teel, P. D. (1991). Off-host physiological ecology of ixodid ticks.Annu. Rev. Entomol. 36, 659–681.

Pal, U., Li, X., Wang, T., Montgomery, R. R., Ramamoorthi, N., Desilva, A. M., Bao, F.,Yang, X., Pypaert, M., Pradhan, D., Kantor, F. S., Telford, S., Anderson, J. F., andFikrig, E. (2004). TROSPA, an Ixodes scapularis receptor for Borrelia burgdorferi. Cell 119,457–468.

Raikhel, A. S. (1983). The intestine. In: Balashov, Y. S. (Ed.), An atlas of ixodid tickultrastructure. Special publication of the Entomological Society of America, Englishtranslation (in Russian, 1979).

Scott, R. C., Schuldiner, O., and Neufeld, T. P. (2004). Role and regulation of starvation-induced autophagy in the Drosophila fat body. Dev. Cell 7, 167–178.

Sonenshine, D. E. (1991). Biology of ticks. Vol. 1. Oxford University Press, New York.Tanaka, M., Liao, M., Zhou, J., Nishikawa, Y., Xuan, X., and Fujisaki, K. (2007).

Molecular cloning of two caspase-like genes from the hard tick Haemaphysalis longicornis.J. Vet. Med. Sci. 69, 85–90.

Tarnowski, B. I., and Coons, L. B. (1989). Ultrastructure of the midgut and blood mealdigestion in the adult tick Dermacentor variabilis. Exp. Appl. Acarol. 6, 263–289.

Umemiya, R., Hatta, T., Liao, M., Tanaka, M., Zhou, J., Inoue, N., and Fujisaki, K.(2007a). Haemaphysalis longicornis: molecular characterization of a homologue of themacrophage migration inhibitory factor from the partially fed ticks. Exp. Parasitol. 115,135–142.

Umemiya, R., Matsuo, T., Hatta, T., Sakakibara, S., Boldbaatar, D., and Fujisaki, K.(2007b). Cloning and characterization of an autophagy-related gene, ATG12, from thethree-host tick Haemaphysalis longicornis. Insect Biochem. Mol. Biol. 37, 975–984.

Umemiya, R., Matsuo, T., Hatta, T., Sakakibara, S., Boldbaatar, D., and Fujisaki, K. (2008).Autophagy-related genes from a tick, Haemaphysalis longicornis. Autophagy 4, 79–81.

Walker, A. R., and Fletcher, J. D. (1987). Histology of digestion in nymphs of Rhipicephalusappendiculatus fed on rabbits and cattle naı̈ve and resistant to the ticks. Int. J. Parasitol. 17,1393–1411.

Williams, J. P., Barker, D. M., Sauer, J. R., Hair, J. A., Ownby, C., and Koch, H. (1985).Ultrastructural changes in the midgut epithelium of unfed lone star ticks with increasingage. Ann. Entomol. Soc. Am. 78, 62–69.

Zhou, J., Liao, M., Hatta, T., Tanaka, M., Xuan, X., and Fujisaki, K. (2006). Identificationof a follistatin-related protein from the tick Haemaphysalis longicornis and its effect on tickoviposition. Gene 372, 191–198.

Zhou, J., Liao, M., Ueda, M., Gong, H., Xuan, X., and Fujisaki, K. (2007). Sequencecharacterization and expression patterns of two defensin-like antimicrobial peptides fromthe tick Haemaphysalis longicornis. Peptides 28, 1304–1310.