Proteomic analysis of differentially expressed proteins in the marine fish parasitic ciliate Cryptocaryon irritans

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Proteomic analysis of differentially expressed proteins in the marine fish parasitic ciliate Cryptocaryon irritans Yong-Zhan Mai a , Yan-Wei Li a , Rui-Jun Li a , Wei Li a , Xia-Zi Huang a , Ze-Quan Mo b , An-Xing Li a,∗ a State Key Laboratory of Biocontrol, Guangdong Province Key Laboratory for Aquatic Economic Animals, School of Life Sciences, Sun Yat-Sen University, Guangzhou, China b College of Animal Science, South China Agricultural University, Guangzhou, China

a r t i c l e

i n f o

Article history: Received 3 December 2014 Received in revised form 26 March 2015 Accepted 7 May 2015 Keywords: Cryptocaryon irritans Comparative proteomics Immunoproteomics

a b s t r a c t Cryptocaryoniasis is a severe disease of farmed marine fish caused by the parasitic ciliate Cryptocaryon irritans. This disease can lead to considerable economic loss, but studies on proteins linked to disease development and antigenic proteins for vaccine development have been relatively scarce to date. In this study, 53 protein spots with differential abundance, representing 12 proteins, were identified based on a pair-wise comparison among theronts, trophonts, and tomonts. Meanwhile, 33 protein spots that elicited serological responses in rabbits were identified, representing 9 proteins. In addition, 27 common antigenic protein spots reacted with grouper anti-sera, representing 10 proteins. Most of the identified proteins were involved in cytoskeletal and metabolic pathways. Among these proteins, actin and ␣tubulin appeared in all three developmental stages with differences in molecular weights and isoelectric points; 4 proteins (vacuolar ATP synthase catalytic subunit ␣, mcm2-3-5 family protein, 26S proteasome subunit P45 family protein and dnaK protein) were highly expressed only in theronts; while protein kinase domain containing protein and heat shock protein 70 showed high levels of expression only in trophonts and tomonts, respectively. Moreover, actin was co-detected with 3 rabbit anti-sera while ␤tubulin, V-type ATPase ␣ subunit family protein, heat shock protein 70, mitochondrial-type hsp70, and dnaK proteins showed immunoreactivity with corresponding rabbit anti-sera in theronts, trophonts, and tomonts. Furthermore, ␤-tubulin, the metabolic-related protein enolase, NADH-ubiquinone oxidoreductase 75 kDa subunit, malate dehydrogenase, as well as polypyrimidine tract-binding protein, glutamine synthetase, protein kinase domain containing protein, TNFR/NGFR cysteine-rich region family protein, and vacuolar ATP synthase catalytic subunit ␣, were commonly detected by grouper anti-sera. Therefore, these findings could contribute to an understanding of the differences in gene expression and phenotypes among the different stages of parasitic infection, and might be considered as a source of candidate proteins for disease diagnosis and vaccine development. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Cryptocaryon irritans is a globally-distributed pathogenic parasitic ciliate that can infect almost all of the ocean teleost fish. C. irritans infection causes severe cryptocaryoniasis and results in mass economic losses for the aquaculture industry. Fish mortality caused by C. irritans infection is increasing over time (Burgess and Matthews, 1994; Colorni and Burgess, 1997). In South China,

∗ Corresponding author at: School of Life Sciences, Sun Yat-sen University, 135 Xingang Wes Street, Haizhu District, Guangzhou 510275, China. Tel.: +86 20 84115113; fax: +86 20 84115113. E-mail address: [email protected] (A.-X. Li).

C. irritans has afflicted farmed fish including grouper, sea bream, yellow croaker, pompano, and the direct economic loss due to cryptocaryoniasis has amounted to over one hundred million yuan RMB (equivalent to about 16 million US dollars) just in the Guangdong Province each year (data not published). Diversified treatments are available currently for the control of cryptocaryoniasis, such as the use of chemotherapeutic agents, fresh water soaking, and the cleaning of used equipment (Cardeilhac and Whitaker, 1988; Colorni and Burgess, 1997; Matthews et al., 1993; Rigos et al., 2013; Yoshinaga, 2001). However, these methods are not suitable for extensive use because of their drawbacks, such as drug residues, time-consumption, and high cost.

http://dx.doi.org/10.1016/j.vetpar.2015.05.004 0304-4017/© 2015 Elsevier B.V. All rights reserved.

Please cite this article in press as: Mai, Y.-Z., et al., Proteomic analysis of differentially expressed proteins in the marine fish parasitic ciliate Cryptocaryon irritans. Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2015.05.004

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C. irritans is a complex unicellular eukaryote that has several distinct developmental stages in its life cycle: the infective theront, the parasitic trophont, and the reproductive tomont (Bai et al., 2008). Many types of marine fish have high mortality rates when infected by theronts, and the development of the disease is associated with the biological progress in the life cycle of C. irritans. Immunoprophylaxis is considered an alternative prevention for cryptocaryoniasis. A vaccine prepared by theronts was utilized to prevent outbreaks of cryptocaryoniasis (Luo et al., 2008; Yambot and Song, 2006), but it is impractical to use this method in largescale operations, since C. irritans cannot be cultured in vitro and the passage of the parasite continuously in fish is time-consuming. An immobilization antigen (i-antigen) isolated from the surface of theronts was identified as a glycosyl phosphatidyl inositol (GPI)anchored surface membrane protein and considered a potential subunit vaccine antigen candidate (Clark et al., 2001; Hatanaka et al., 2008). Additionally, a modified DNA vaccine based on an i-antigen sequence was demonstrated to protect fish against infection by C. irritans (Priya et al., 2012). Furthermore, cross-protection was elicited in Mozambique tilapia against two different immobilization serotypes of C. irritans, indicating that other candidate components could be applied to vaccine development (Misumi et al., 2011). Proteomic approaches are available to identify proteins involved in complex biological progress and allow for large-scale screening of potential vaccine candidate proteins from parasites. For example, serine proteases, DNase II and trypsin were identified as the early specific diagnostic antigens of trichinellosis by twodimensional electrophoresis (2-DE) and Western blot (Wang et al., 2014). Different strains of Trypanosoma cruzi zymodeme were analyzed by 2-DE and cruzipain was identified as an important antigen among these proteins from T. cruzi (Kikuchi et al., 2010). In this study, we performed differential proteomics and immunoproteomics approaches to identify differentially expressed proteins in three developmental stages, and immunogenic proteins were recognized by rabbit and grouper anti-sera. This study laid the foundation for the discovery of therapeutic targets and the development of vaccine against C. irritans. 2. Materials and methods 2.1. Parasite strain identification and collection C. irritans, in this study, was isolated from naturally infected Trachinotus ovatus in Daya Bay Guangdong Province, China, and were propagated using T. ovatus as the host according to a previously described procedure (Dan et al., 2006). Trophonts were gently gathered from the bottom of the special collection unit as soon as they detached from infected fish. After careful cleaning, trophonts were transferred to flasks and allowed to transform into tomonts for 6–8 h. 2–3 Days later, theronts were released from tomont cysts. Formalin fixed theronts, trophonts, and tomonts were collected and centrifuged for 5 min at 8000 rpm and pellets were immediately frozen in liquid nitrogen. 2.2. Preparation of rabbit anti-C. irritans sera The collected theronts, trophonts, and tomonts were separately sonicated on ice (80 W, 1 s pulses, 1 s pause, 10 times, with 2 min intervals) and adjusted to 2 mg protein ml−1 with phosphatebuffered saline (pH 7.4, PBS) for vaccination (Zhou et al., 2014; Xu et al., 2009). To prepare rabbit anti-C. irritans sera, New Zealand rabbits were immunized by subcutaneous injection with 1 mg protein from sonicated theronts, trophonts, or tomonts. The rabbits were boost-immunized twice with half of the amount of the

corresponding proteins at 1-week intervals. One week after the last immunization, the blood of the rabbits was collected individually; serum was separated from the blood and stored at –80 ◦ C until use. The control serum was collected from corresponding pre-immunized rabbits individually. 2.3. Preparation of grouper (Epinephelus coioides) anti-C. irritans sera To prepare grouper anti-C. irritans serum, 30 grouper were divided into two groups and cultured at 25 ± 3 ◦ C. One group (infection group) was challenged by infection with live theronts (about 30,000 theronts per fish) once weekly over 4 weeks, and the other group (immunization group) was vaccinated by intraperitoneal injection with proteins isolated from formalin fixed theronts (proteins were sampled as described in Section 2.2). The grouper in the immunization group were boosted once by immunization with half the amount of proteins 2 weeks after the initial immunization. One week after the last infection or vaccination, the blood was collected, and serum was separated from the blood and stored at −80 ◦ C until use. The control serum was collected under the same condition from grouper that were not infected with live theronts or immunized with formalin fixed theronts. 2.4. Preparation of anti-grouper immunoglobulin M (IgM) serum Grouper IgM was purified according to a previous study with some modification (Cheng et al., 2006). Briefly, 5 ml of Protein G Sepharose (GE Health) was packed into 10 cm × 1 cm column and equilibrated with 10 column volumes of PBS. The grouper serum was filtered through a filter with 0.22 mm pore size, and added onto the resin at a 1:5 dilution with PBS and flowed through the packed column at 4 ◦ C for a minimum of five times. The column was washed with 10 column volumes of PBS, and the protein was eluted from the column with an eluent solution (50 mM glycine, pH 2.7) and distributed into a sterilized centrifuge tube containing 1/9 eluent volume of neutral buffer (1 M Tris–base, 1.5 M NaCl, 1 mM EDTA, 0.5% sodium azide, pH 8.0). The collected eluate containing grouper IgM was concentrated by ultrafiltration (Amicon Ultra 10K NMWL device, Millipore) and the retained fluid was subjected to SDS-PAGE. The gel was stained by Coomassie Brilliant Blue R-250, and the protein band from grouper IgM heavy chains was excised and confirmed by using an ABI 4800 MALDI-TOF/TOF proteomics analyzer mass spectrometer (Applied Biosystems, USA). The purified IgM was injected subcutaneously into a New Zealand rabbit and anti-serum was prepared as described in Section 2.2. 2.5. Protein sample preparation Approximately 100 mg of parasite sample was ground into powder under liquid nitrogen and homogenized in 1 ml lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, and 1 mM PMSF). The mixture was intermittently sonicated on ice and centrifuged at 12,000 rpm for 20 min. The supernatant was collected and four volumes of ice-cold acetone were added. The solution was incubated at −20 ◦ C for 2 h and centrifuged at 12,000 rpm for 20 min. The protein precipitate was air-dried and then re-suspended in 200 ␮l lysis buffer (7 M urea, 2 M thiourea, and 4% CHAPS). The protein concentration was determined with a Bradford assay against a bovine serum albumin (BSA) standard curve and the concentration was adjusted to 5 ␮g/␮l for storage at −80 ◦ C until use. 2.6. 2-D gel electrophoresis The 2-DE was carried out according to a previously described study (Li et al., 2014). 500 ␮g of the protein sample was mixed with

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Fig. 1. Representative 2-DE gels of three development stages among theronts, trophonts, and tomonts of Cryptocaryon irritans. Proteins (500 ␮g) were separated with 17 cm IPG strips on a non-linear pH range of 4–7 and visualized with Coomassie brilliant blue G-250 staining. Proteins of theronts (A), trophonts (B), and tomonts (C) were displayed, respectively; proteins spots highly expressed only in theronts (spots 1–26), trophonts (spots 27–38), and tomonts (spots 39–53) were present and listed in Table 1.

rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 1% DTT, 0.5% Bio-Lyte® ampholyte, pH 3–10 NL) to a final volume of 300 ␮l. The mixed rehydration buffer was loaded onto 17 cm ReadyStripTM IPG dried strips (Bio-Rad pH 4–7 NL) and incubated on a focusing plate at 20 ◦ C and 50 V for 12 h. Subsequently, the protein loaded IPG strips were subjected to isoelectrofocusing (IEF) with a PROTEAN® IEF cell (Bio-Rad) and separated under the following conditions: 300 V for 1 h, 700 V for 1 h, 1500 V for 1.5 h; 9000 V for 3 h, and then constant for a total of 45,000 Vh (Liu et al., 2012). After the first dimension of electrophoresis, the IPG strips were equilibrated in the equilibration solution (6 M urea, 2% SDS, 30% glycerol, and 50 mM Tris–HCl (pH 8.8) with 1% DTT) for 15 min, followed by alkylation in the same solution with 2.5% iodoacetamide (IAM) for 15 min (Zhou et al., 2014). The second stage of electrophoresis was performed in a PROTEAN Plus DodecaTM cell (Bio-Rad) and the proteins were separated with 12% hand-made acrylamide gels with 1% agarose sealant by running at 15 mA until the bromophenol blue solvent front traveled to the bottom of the gels. The gels were visualized via Coomassie brilliant blue G-250 staining, imaged on GS-800TM Calibrated Densitometer (Bio-Rad) and analyzed with PDQuestTM v8.0 analysis software (Bio-Rad). All of the significantly changed protein spots, as determined by Student t-test (p < 0.05) were detected automatically following the manual removal of streaks, speckles, and artificial spots, as described in the instruction manuals.

2.7. Detection of immunogenic proteins The immunoproteomics analysis of C. irritans was performed in accordance with a protocol described previously (Liu et al., 2012). Briefly, the proteins extracted from C. irritans and separated by twodimensional electrophoresis were electrophoretically transferred onto a nitrocellulose filter (Pall) in a gel sandwich using TransBlot® Electrophoretic Transfer Cell (Bio-Rad) at 200 mA for 3 h. The NC membrane was blocked with 5% skim milk in TBS overnight at 4 ◦ C and subsequently incubated for 2 h with primary antibodies (grouper anti-C. irritans sera diluted at 1:100 in TBS; rabbit antiC. irritants sera diluted at 1:500 in TBS) and secondary antibodies (rabbit anti-grouper IgM sera diluted at 1:500 in TBS) at room temperature. The immunogenic proteins were visualized by incubating the membrane with horseradish peroxidase-conjugated goat antirabbit IgG antibodies diluted at 1:5000 in TBS, at room temperature for 1 h, and then the membrane was developed with DAB substrate (0.002% DAB, 0.03% H2 O2 ). 2.8. Mass spectrometry analysis Selected proteins spots from C. irritans were excised from the gels and subjected to destaining with 50% methanol, followed by 50% acetonitrile (ACN), and desiccation under vacuum. The dried gel pieces were then rehydrated and incubated in 5 ␮l coverage

Please cite this article in press as: Mai, Y.-Z., et al., Proteomic analysis of differentially expressed proteins in the marine fish parasitic ciliate Cryptocaryon irritans. Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2015.05.004

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4 Table 1 Differentially expressed proteins of Cryptocaryon irritans. Spots no.a

Disease stage

Protein descriptionb

MASCOT scorec

Theoretical Mr d (Da)

1

Theront

67

11,087

2 3 4 5 6 7 8 9

Theront Theront Theront Theront Theront Theront Theront Theront

92 310 123 122 162 68 121 44

10

Theront

11

Theront

12

Theront

13

Theront

14

Theront

15

Theront

16

Theront

17 18 19 20 21 22 23

Theront Theront Theront Theront Theront Theront Theront

24

Theront

25

Theront

26

Theront

Vacuolar ATP synthase catalytic subunit ␣, putative [Ichthyophthirius multifiliis] ␤-Tubulin [Paramecium caudatum] ␤2-Tubulin [Euplotes octocarinatus] ␣-Tubulin [Tokophrya lemnarum] ␣-Tubulin [Tokophrya lemnarum] ␣-Tubulin [Tokophrya lemnarum] ␣-Tubulin [Tokophrya lemnarum] ␣-Tubulin [Tokophrya lemnarum] Enolase [Paramecium multimicronucleatum] mcm2-3-5 family protein, putative [Ichthyophthirius multifiliis] Hypothetical protein [Paramecium tetraurelia strain d4-2] Hypothetical protein [Paramecium tetraurelia strain d4-2] 26S Proteasome subunit P45 family protein [Tetrahymena thermophila] Unnamed protein product [Paramecium tetraurelia] dnaK protein [Tetrahymena thermophila] dnaK protein [Tetrahymena thermophila] ␤-Tubulin [Paramecium caudatum] ␤-Tubulin T2 [Euplotes focardii] ␤-Tubulin [Paramecium caudatum] Actin [Cryptocaryon irritans] ␤-Tubulin ␤2-Tubulin [Euplotes octocarinatus] Hypothetical protein IMG5 173650 [Ichthyophthirius multifiliis] Hypothetical protein IMG5 192870 [Ichthyophthirius multifiliis] Unnamed protein product [Paramecium tetraurelia] Hypothetical protein IMG5 015480 [Ichthyophthirius multifiliis]

27 28 29 30 31 32 33

Trophont Trophont Trophont Trophont Trophont Trophont Trophont

34 35

Trophont Trophont

36

Trophont

37 38

Trophont Trophont

39

Tomont

40

Tomont

41

Tomont

42 43 44

Tomont Tomont Tomont

45

Tomont

46

Tomont

47

Tomont

48

Tomont

Enolase [Oxytricha trifallax] Actin [Metopus es] Actin [Metopus es] ␣-Tubulin [Strombidinopsis sp.] Actin [Metopus palaeformis] Actin [Metopus es] Hypothetical protein IMG5 155090 [Ichthyophthirius multifiliis] Actin [Nyctotherus ovalis] Unnamed protein product [Paramecium tetraurelia] Unnamed protein product [Paramecium tetraurelia] Actin [Nyctotherus ovalis] Protein kinase domain containing protein [Oxytricha trifallax] Hypothetical protein [Paramecium tetraurelia strain d4-2] Unnamed protein product [Paramecium tetraurelia] Unnamed protein product [Paramecium tetraurelia] ␣-Tubulin [Tokophrya lemnarum] Actin [Metopus palaeformis] ␤-Tubulin, putative [Ichthyophthirius multifiliis] Hypothetical protein OXYTRI 07847 [Oxytricha trifallax] Heat shock protein 70 [Lamtostyla sp. LPJ-2005] ␤-Tubulin [Monosiga brevicollis MX1] ␤-Tubulin

Theoretical pIe

Peptides matched

Protein IDf

4.89

4

gi|471219829

14,415 50,087 40,806 40,806 40,806 40,806 40,806 38,617

5.23 4.84 6.54 6.54 6.54 6.54 6.54 5.94

4 13 8 8 8 8 8 1

gi|15212109 gi|112383583 gi|21632080 gi|21632080 gi|21632080 gi|21632080 gi|21632080 gi|15667715

15

19,413

5.89

1

gi|471228361

18

17,772

9.73

1

gi|145515693

238

19,030

7.78

6

gi|145483021

18

18,422

9.1

1

gi|146171839

64

57,340

6.6

9

gi|124417901

52

17,244

5.74

1

gi|118358030

29

17,244

5.74

1

gi|118358030

86 166 71 189 73 106 84

14,415 50,159 14,415 41,915 49,937 50,087 27,958

5.23 4.81 5.23 5.5 4.82 4.84 4.92

4 12 4 7 8 7 7

gi|15212109 gi|224712111 gi|15212109 gi|359744459 gi|417854 gi|112383583 gi|471221868

44

11,809

6.3

1

gi|471220004

34

82,373

4.86

1

gi|124405526

34

59,681

8.73

1

gi|340508687

109 146 112 87 146 68 67

50,472 29,696 29,696 39,806 31,464 29,696 27,844

7.7 5.23 5.23 6.01 5.16 5.23 5.02

4 5 4 6 6 6 7

gi|403364642 gi|110645076 gi|110645076 gi|23957245 gi|160337395 gi|110645076 gi|471223876

103 62

29,970 110,506

5.23 5.4

4 9

gi|110645001 gi|124419106

62

75,071

5.86

10

gi|124397486

91 30

29,970 45,479

5.23 9.07

3 1

gi|110645001 gi|403368758

16

12,386

10.19

1

gi145515187

62

13,528

10.38

4

gi|124429727

66

98,883

6.03

9

gi|124391475

82 120 59

40,806 31,464 50,037

6.54 5.16 4.81

7 7 5

gi|21632080 gi|160337395 gi|471233184

58

47,649

6.24

7

gi|403343894

77

40,567

5.89

5

gi|67462298

71

21,934

5.14

4

gi163778536

385

49,937

4.82

14

gi|417854

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Table 1 (Continued) Spots no.a

Disease stage

Protein descriptionb

MASCOT scorec

Theoretical Mr d (Da)

49

Tomont

58

18,816

50

Tomont

65

51 52 53

Tomont Tomont Tomont

Hypothetical protein OXYTRI 21868 [Oxytricha trifallax] Heat shock protein 70 [Lamtostyla sp. LPJ-2005] Actin [Nyctotherus ovalis] Actin [Nyctotherus ovalis] Hypothetical protein TTHERM 00658990 [Tetrahymena thermophila SB210]

72 101 65

a b c d e f

Theoretical pIe

Peptides matched

Protein IDf

5.77

4

gi|403362054

40,567

5.89

6

gi|67462298

29,970 29,970 13829

5.23 5.23 6.56

3 3 5

gi|110645001 gi|110645001 gi|89305867

Protein spots number in Fig. 1. Protein description in NCBI proteins database. MASCOT scores. Theoretical molecular weights. Theoretical isoelectric point. Accession number in NCBI database.

solution (10% ACN, 50 mM NH4 HCO3 ) containing 10 ng trypsin (Promega) for 16 h at 37 ◦ C. Next, the extracted peptides were dissolved in 1.5 ␮l of resolution solution (30% ACN and 0.1% trifluoroacetic acid (TFA)), and 0.8 ␮l of this mixture was loaded onto a steel target plate with 0.5 ␮l ␣-cyano-4-hydroxycinnamic acid (CHCA) matrix (5 mg/ml, dissolved in 50% ACN with 0.1% TFA). The mass spectrometric analysis was performed on an ABI 4800 MALDI-TOF/TOF mass spectrometer (Applied Biosystems, USA) and parameters were set similarly to previous protocols (Zhou et al., 2014). Data were searched against the Mascot database, with error tolerances of 50 ppm and 50 ppm for MS and MS/MS, respectively. One missed cleavage was allowed; carbamidomethy and oxsidation modifications were selected. The criteria for successfully identified proteins were one or more tryptic peptide matches to the protein sequence and at least one peptide with p < 0.05.

3. Results 3.1. Identification of C. irritans differentially expressed proteins To identify differentially expressed proteins in theronts, trophonts, and tomonts, 2-DE based on a pair-wise comparison was performed. According to the previous study, the comparative analyses of theronts, trophonts, and tomonts were conducted in a pairwise fashion and 83 protein spots displayed significant changes of more than 3 fold between samples (Zhou et al., 2014). Of these, 53 protein spots were successfully detected, including 26 theront protein spots, 12 trophont protein spots, and 15 tomont protein spots, respectively. These 53 protein spots represented 12 proteins of C. irritans, including ␣-tubulin, ␤-tubulin, actin, enolase, dnak, vacuolar ATP synthase catalytic subunit ␣, mcm2-3-5 family proteins, 26S proteasome subunit P45 family protein, protein kinase domain containing protein, and heat shock protein 70 (Table 1). A large proportion of proteins showing differential expression were homologous to ␣-tubulin and actin. The spot 9 in theront (Fig. 1A) and spot 27 in trophont (Fig. 1B), were identified as enolase with different isoelectric points (pI) and relative molecular weight (Mr) values, while ␤-tubulin appeared in theronts and tomonts without detection in trophonts. The other identified proteins were present in only one developmental stage. Vacuolar ATP synthase catalytic subunit ␣, 26S proteasome subunit P45 family protein, mcm2-35 family protein, and dnak protein were identified in theronts, whereas, protein kinase domain containing protein and heat shock protein 70 were detected in trophonts and tomonts, respectively. The proteins expressed at a high level in theront were mainly ␣tubulin, showing some fluctuation of pI values, which were present as a train of spots 4–6 (Fig. 1A). The proteins that were more abundant in theront than tomonts were also mainly a structural protein,

␤-tubulin, with some heterogeneity in terms of pI and Mr values, appearing as spots 17–19 and 22 (Fig. 1A). 3.2. Identification of C. irritans immunogenic proteins with rabbit anti-sera To identify C. irritans immunogenic components that can induce organism antibody responses, proteins of theronts, trophonts, and tomonts were subjected to 2-DE coupled with immunoblotting with corresponding rabbit anti-sera, and 33 protein spots were specially recognized, representing 9 proteins. Of these, 10 protein spots in theronts (Fig. 2A), 12 protein spots in trophonts (Fig. 2B), and 11 protein spots in tomonts (Fig. 2C) were immunoreactive with corresponding rabbit anti-sera. As summarized in Table 2, the majority of proteins were characterized as structural proteins, such as actin and tubulin. Among these identified proteins, actin was recognized by all rabbit anti-sera against theronts, trophonts, and tomonts, while the other six proteins were recognized by only one rabbit anti-sera each: ␣-tubulin and ␤-tubulin by rabbit antitheront sera, V-type ATPase ␣ subunit family protein, heat shock protein 70 and mitochondrial-type hsp70 by rabbit anti-trophont sera, and dnak by rabbit anti-tomont sera. Meanwhile, numerous hypothetical proteins with uncharacterized function reacted with rabbit anti-sera against C. irritans. Although most proteins spots were parallel to their respective molecular masses and isoelectric points on the 2-D gels it is worth noting discrepancies in the theoretical molecular mass of some proteins. For example, spot 6 and 7 shown in Fig. 2A, were both confirmed as ␤-tubulin and their predicted molecular weights were less than that observed in the 2-DE gel. Whereas, in Fig. 2B, the visible molecular mass of the spot 20, the V-type ATPase, displayed deviation from the theoretical calculated molecular weight value, which may indicate a post-translational modification, or different forms existing in nature. 3.3. Identification of C. irritans immunogenic proteins with grouper anti-sera As shown in Fig. 3, a total of 27 protein spots were immunoreactive with grouper anti-theront serum, representing 10 proteins. Among these protein spots, 9 spots named as 1, 3–5, 7–9, 11, 23 were characterized by mass spectrometry as ␤-tubulin, 7 spots termed as 2, 13, 16, 17, 20, 21, 26 were identified as hypothetical proteins, 2 spots marked as 15 and 19 were confirmed as protein kinase domain containing protein, and 7 spots recognized by grouper anti-sera, such as spot 6, 10, 12, 14, 22, 25, 27, were confirmed as polypyrimidine tract-binding protein, NADHubiquinone oxidoreductase 75 kDa subunit, glutamine synthetase, enolase, malate dehydrogenase, TNFR/NGFR cysteine-rich region

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6

Fig. 2. Immunogenic proteins of Cryptocaryon irritans recognized by rabbit anti-serum. (A) Immunogenic proteins of theronts detected by rabbit anti-sera; (B) immunogenic proteins of trophont detected by rabbit anti-sera; (C) immunogenic proteins of tomont detected by rabbit anti-sera. Immunogenic proteins of theronts (spots 1–10), trophonts (spots 11–22), and tomonts (spots 23–33) were listed in Table 2.

family protein, and vacuolar ATP synthase catalytic subunit, respectively (Table 3). The potential antigens inducing fish immune responses were co-detectable with grouper anti-sera by infection and immunization (Fig. 3A and B). Immunoreactive protein spots of theronts were clearly visible with approximately equal numbers of reaction spots whether the development of grouper anti-sera was performed by infection or immunization. Compared to Fig. 2A, 27 protein spots by co-detection with two kinds of grouper anti-sera were obvious in concentrating distribution with molecular mass values between 35 kDa and 55 kDa. Similarly, 10 reactive spots of theronts with rabbit anti-sera were apparent in contiguous distribution with molecular weights between 36 kDa and 95 kDa. 4. Discussion In recent years, an increasing number of cryptocaryoniasis outbreaks have led to substantial economic loss in fisheries and aquaculture (Luo et al., 2008; Sun et al., 2006). Immunoprophylaxis is considered as an effective and efficient approach to control this disease, for the fact that the antigens of C. irritans can induce a strong immune response involving the generation of specific antibodies and may be utilized as therapeutic targets for cryptocaryoniasis (Bai et al., 2008; Luo et al., 2007; Yambot and Song, 2006). Therefore, it is necessary to screen potential antigens of C. irritans. In this study, a total of 12 differentially expressed proteins

and 16 immunogenic proteins were identified, of which the vast majority were involved in various biological functions including cytoskeleton structure, metabolism, stress response, signal transduction, and regulation. The comparative proteomic profiles of C. irritans showed these proteins to be either significantly increased or decreased in abundance during the different developmental stages. This proteomic approach contributes to screen new antigens for parasitic diseases and research on the pathogenic mechanism of parasites. In this study, the immunogenic proteins recognized by rabbit and grouper anti-sera covered all 12 differentially expressed proteins, indicating these proteins in different abundance might be pursued as targets for therapeutic vaccines. Thus, the predominance of actin and ␣-tubulin present in all stages renders them ideal candidate proteins for vaccine trials and development. Enolase was identified in theronts and trophonts, ␤-tubulin in theronts and tomonts, suggesting that these two proteins were also involved in parasite growth and development and might be exploited as immunodiagnostic antigens for the control of cryptocaryoniasis. Immunoproteomics analysis was also used to identify as many immunogenic proteins as possible. In the immunoblot images of theront proteins, the antigens recognized with rabbit anti-sera were different from those with grouper anti-sera: 5 distinct proteins were detected by rabbit anti-theront sera, while 10 proteins were recognized by grouper anti-theronts sera. This result indicated the immune responses stimulated by parasitic antigens

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Table 2 Immunogenic proteins of Cryptocaryon irritans identified with rabbit anti-sera. Spots no.a

Disease stage

Protein descriptionb

MASCOT scorec

1 2 3

Theront Theront Theront

233 218 34

4

Theront

5 6 7 8

Theront Theront Theront Theront

9 10

Theront Theront

Actin [Cryptocaryon irritans] ␤-Tublin [Euplotes raikovi] Hypothetical protein TTHERM 00388280 [Tetrahymena thermophila SB210] Unnamed protein product [Paramecium tetraurelia] ␤2-Tubulin [Euplotes octocarinatus] ␤-Tubulin chain ␤-Tubulin chain Unnamed protein product [Paramecium tetraurelia] ␤-Tubulin chain ␣-Tubulin [Tokophrya lemnarum]

11

Trophont

12

Trophont

13 14

Trophont Trophont

15

Trophont

16

Trophont

17 18

Trophont Trophont

19

Trophont

20

Trophont

21

Trophont

22

Trophont

23

Tomont

24

Tomont

25

Tomont

26

Tomont

27 28

Tomont Tomont

29

Tomont

30

Tomont

31

Tomont

32

Tomont

33

Tomont

a b c d e f

Hypothetical protein OXYTRI 13090 [Oxytricha trifallax] Hypothetical protein TTHERM 00123730 [Tetrahymena thermophila SB210] Actin [Metopus palaeformis] Unnamed protein product [Paramecium tetraurelia] Unnamed protein product [Paramecium tetraurelia] Hypothetical protein TTHERM 00123730 [Tetrahymena thermophila SB210] Actin [Metopus es] Hypothetical protein OXYTRI 11328 [Oxytricha trifallax] Heat shock protein 70 [Lamtostyla sp. LPJ-2005] V-type ATPase, A subunit family protein [Tetrahymena thermophila] Mitochondrial-type hsp70 1 [Paramecium caudatum] Unnamed protein product [Paramecium tetraurelia] Hypothetical protein IMG5 015480 [Ichthyophthirius multifiliis] Hypothetical protein IMG5 015480 [Ichthyophthirius multifiliis] Hypothetical protein TTHERM 00123730 [Tetrahymena thermophila SB210] Hypothetical protein IMG5 007980, partial [Ichthyophthirius multifiliis] Actin [Metopus es] Hypothetical protein OXYTRI 18482 [Oxytricha trifallax] Hypothetical protein IMG5 107510 [Ichthyophthirius multifiliis] Unnamed protein product [Paramecium tetraurelia] Hypothetical protein IMG5 091060 [Ichthyophthirius multifiliis] dnaK protein [Tetrahymena thermophila] Hypothetical protein TTHERM 00568030 [Tetrahymena thermophila SB210]

Theoretical Mr d (Da)

Theoretical pIe

Peptides matched

Protein IDf

41,915 50,060 227,392

5.5 4.77 8.68

9 7 2

gi|359744459 gi|224712109 gi|89301099

39

29,795

9.40

1

gi|124406073

72 128 356 43

50,087 49,986 49,986 113,608

4.84 4.81 4.81 5.9

9 8 12 1

gi|112383583 gi|586077 gi|586077 gi|124414879

433 296

49,937 40,813

4.82 6.1

11 12

gi|417854 gi|21632075

37

158,718

9.27

1

gi|403334904

39

109,236

6.02

1

gi|89292635

148 33

31,464 56,075

5.16 8.12

6 1

gi|160337395 gi|124407581

47

82,373

4.86

1

gi|124405526

37

109,236

6.02

1

gi|89292635

79 71

29,696 111,111

5.23 5.51

3 14

93

40,567

5.89

3

gi|67462298

101

69,606

6.33

3

gi|146170652

171

49,076

8.41

4

gi|315661097

40

72,100

8.03

2

gi|124423714

32

59,681

8.73

1

gi|340508687

32

59,681

8.73

1

gi|340508687

38

109,236

6.02

1

gi|89292635

20

13,999

8.49

1

gi|471235982

69 28

29,696 94,994

5.23 7.43

2 1

gi|110645076 gi|403367914

67

61,973

5.55

4

gi|471228265

65

46,331

9.65

10

gi|124398136

117

82,283

5.19

8

gi|471229810

70

17,244

5.74

2

gi|118358030

41

30372

6.23

1

gi|225566845

gi|110645076 gi|403337865

Protein spots number in Fig. 2. Protein description in NCBI proteins database. MASCOT scores. Theoretical molecular weights. Theoretical isoelectric point. Accession number in NCBI database.

depend on host specificity. C. irritans predominantly infects marine fish species rather than rabbit, mouse or any other mammal, and the immune responses of fish generated from infection by C. irritans are stronger in intensity and of longer duration than those of other animal against heterologous protein extracted from C. irritans (Xu

et al., 2008). Meanwhile, actin showed common immunogenicity in all three rabbit anti-sera, and ␤-tubulin of theronts was immunoreactive with rabbit and grouper anti-sera, suggesting that these two proteins might be strongly reactive to the host immune system. In addition, we performed 2-DE combined with immunoblotting by

Please cite this article in press as: Mai, Y.-Z., et al., Proteomic analysis of differentially expressed proteins in the marine fish parasitic ciliate Cryptocaryon irritans. Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2015.05.004

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Fig. 3. Immunogenic proteins of Cryptocaryon irritans recognized by grouper anti-serum. Immunogenic proteins of theronts recognized by anti-sera from grouper infected with theronts (A) and immunized with theronts proteins (B) were displayed. Immunogenic proteins co-detected by these two types of sera (spots 1–27) were listed in Table 3.

grouper anti-theront sera, but not grouper anti-trophont or antitomont sera, given that only infective theronts invade the gills and skin of fish and develop into parasitic trophonts on the surface of fish. Of these identified protein spots, ␤-tubulin was detected in differentially expressed protein profiles and immunoproteomics profiles with rabbit and grouper anti-sera. Another member of the tubulin superfamily, ␣-tubulin, also appeared in all three developmental stages and was recognized by rabbit anti-sera. However, it was not detected with grouper anti-sera. Tubulins are highlyconserved proteins shared by all types of eukaryotes and are responsible for polymerization of microtubules involved in cell movement and migration (Janke and Kneussel, 2010). There was no evidence that tubulins of ciliates were involved in the host immune response (Libusova and Draber, 2006). However, previous studies have demonstrated that tubulins of other parasitic protozoan are of

certain value in clinical application because of its immunogenicity. Immunization with ␣-tubulin-GST protein conferred e partial protection in mice against Eimeria acervulina oocyst challenge (Ding et al., 2008). A DNA vaccine containing the full length ␤-tubulin gene of Trypanosoma evansi was utilized to inoculate mice to elicit specific humoral immune responses, and after a lethal challenge of T. evansi bloodstream stage trypomastigotes, prolonged survival was observed in mice immunized with this DNA vaccine (Kurup and Tewari, 2012). Meanwhile, this DNA vaccine based on T. evansi ␤-tubulin gene also protected the immunized mice from lethal challenges from other species of trypanosoma (Li et al., 2007). Another important immunogenic cytoskeletal protein identified in this study was actin. In this study, actin appeared in all three developmental stages and was commonly recognized by rabbit anti-sera. Previous studies of parasitic protozoan reported that actin was involved in the invasion into host cells and the migration

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Table 3 Immunogenic proteins of Cryptocaryon irritans identified with grouper anti-sera. Spots no.a

Disease stage

Protein descriptionb

MASCOT scorec

1 2

Theront Theront

310 238

50,087 19,030

4.84 7.78

13 6

gi|112383583 gi|145483241

3 4 5 6

Theront Theront Theront Theront

62 72 121 29

14,415 14,415 14,415 15,091

5.23 5.23 5.23 9.66

3 4 7 4

gi|15212109 gi|15212109 gi|15212109 gi|118399788

7 8 9 10

Theront Theront Theront Theront

121 29 71 37

14,415 14,415 14,415 28,734

5.23 5.23 5.23 6.52

7 2 4 5

gi|15212109 gi|15212109 gi|15212109 gi|229595475

11 12

Theront Theront

131 97

14,415 12,690

5.23 6.34

7 5

gi|15212109 gi|118396127

13

Theront

238

19,030

7.78

6

gi|145483241

14

Theront

44

38,617

5.94

1

gi|15667715

15

Theront

30

12,532

4.59

3

gi|146179021

16

Theront

30

12,393

10.17

3

gi|145516022

17

Theront

25

11,906

4.35

3

gi|145483241

18

Theront

34

18,338

9.67

4

gi|118400437

19

Theront

29

12,532

4.59

4

gi|146179021

20

Theront

42

11,906

4.35

4

gi|145483241

21

Theront

43

11,906

4.35

3

gi|145483241

22

Theront

46

14,521

6.33

4

gi|471229898

23 24

Theront Theront

56 30

14,415 13,646

5.23 6.56

3 2

gi|15212109 gi|471227499

25

Theront

31

12,836

10.31

3

gi|118364137

26

Theront

28

13,926

8.49

3

gi|145518047

27

Theront

␤2-Tubulin [Euplotes octocarinatus] Hypothetical protein [Paramecium tetraurelia strain d4-2] ␤-Tubulin [Paramecium caudatum] ␤-Tubulin [Paramecium caudatum] ␤-Tubulin [Paramecium caudatum] Polypyrimidine tract-binding protein [Tetrahymena thermophila] ␤-Tubulin [Paramecium caudatum] ␤-Tubulin [Paramecium caudatum] ␤-Tubulin [Paramecium caudatum] NADH-ubiquinone oxidoreductase 75 kDa subunit, mitochondrial precursor, putative [Tetrahymena thermophila] ␤-Tubulin [Paramecium caudatum] Glutamine synthetase, catalytic domain containing protein [Tetrahymena thermophila] Hypothetical protein [Paramecium tetraurelia strain d4-2] Enolase [Paramecium multimicronucleatum] Protein kinase domain containing protein [Tetrahymena thermophila] Hypothetical protein [Paramecium tetraurelia strain d4-2] Hypothetical protein [Paramecium tetraurelia strain d4-2] Hypothetical protein TTHERM 00600700 [Tetrahymena thermophila] Protein kinase domain containing protein [Tetrahymena thermophila] Hypothetical protein [Paramecium tetraurelia strain d4-2] Hypothetical protein [Paramecium tetraurelia strain d4-2] Malate dehydrogenase, putative [Ichthyophthirius multifiliis] ␤-Tubulin [Paramecium caudatum] Hypothetical protein IMG5 116460 [Ichthyophthirius multifiliis] TNFR/NGFR cysteine-rich region family protein [Tetrahymena thermophila] Hypothetical protein [Paramecium tetraurelia strain d4-2] Vacuolar ATP synthase catalytic subunit a, putative [Ichthyophthirius multifiliis]

83

13,500

4.84

6

gi|471219829

a b c d e f

Theoretical Mr d (Da)

Theoretical pIe

Peptides matched

Protein IDf

Protein spots number in Fig. 3. Protein description in NCBI proteins database. MASCOT scores. Theoretical molecular weights. Theoretical isoelectric point. Accession number in NCBI database.

motility of parasites (Ferreira et al., 2006; Skillman et al., 2012). A DNA vaccine of Trypanosoma gondii actin was able to trigger a strong systemic and mucosal response against T. gondii independently, and the recombinant T. evansi actin could induce protective immunity against infection of four other trypanosomes (Li et al., 2009; Yin et al., 2013). Even though there was no direct proof that actin in ciliates could evoke the host immunity against ciliates infection, mice immunized with a recombinant actin-depolymerizing factor (ADF) of C. irritans produced specific antibodies against this protein (Huang et al., 2013). In addition, tubulin and actin could mediate heterotrimeric G protein signal transduction and result to the interplay of signaling pathways and cytoskeletal dynamics, in which the parasite G proteins-coupled receptors were identified as an antigen against host humoral antibodies (Campos et al., 2014; Schappi et al., 2014). Therefore, actin and tubulin, both of which are present

in C. irritans, might be utilized as targets for vaccine development to control cryptocaryoniasis. The third promising protein in this study was enolase, which was present in the infective theront and parasitic trophont, and immunoreactivity with grouper anti-sera. Enolase is one of limiting enzymes in the process of glycolysis with a highly conserved gene structure. In previous studies, enolase could provide partial protection against parasites in mice immunized with the recombinant protein and the silencing of enolase expression by an RNA interference approach caused delays in infective development (Avilan et al., 2011; Chen et al., 2011, 2012; Wang et al., 2011; Wilson et al., 2004; Yang et al., 2010). In this study, enolase was recognized by the grouper anti-sera, not by sera from rabbit immunized with C. irritans proteins, suggesting that it could be a candidate antigen to immunize fish against cryptocaryoniasis.

Please cite this article in press as: Mai, Y.-Z., et al., Proteomic analysis of differentially expressed proteins in the marine fish parasitic ciliate Cryptocaryon irritans. Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2015.05.004

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The fourth protein identified as a potential candidate antigen was vacuolar ATP synthase catalytic subunit ␣, because it was highly expressed in theronts and detected by rabbit and grouper anti-sera. The main function of vacuolar ATP synthase (V-ATPases) is to pump protons across membranes energized by hydrolysis of ATP to cause limited acidification of the internal membranes and maintain an acidic pH inside the vacuole (Gruber and Marshansky, 2008; Nakanishi-Matsui et al., 2010). The pathogenicity and transition in the life cycle of parasites were associated with the ion transport process dominated by V-ATPases (Lv et al., 2014; Porcel et al., 2000). Recombinant V-ATPase protein of Clonorchis sinensis could be probed by rat anti-serum and C. sinensis-infected human serum in a Western blotting experiment, suggesting its strong antigenicity (Lv et al., 2014). In this study, V-ATPase in theronts with higher expression might be involved in the process of hostinfection and could be used as cryptocaryoniasis vaccines. Heat shock protein 70 (hsp70) in this study might be potential antigens used for vaccine development, even though it showed a stronger reaction with the rabbit anti-sera than fish. However, other studies point to the potential of this protein, as hsp70 of T. gondii is a parasite virulence factor that is expressed during T. gondii stage conversion (Barenco et al., 2014). A previous study revealed that recombinant hsp70 of Leishmania infantum elicited immune responses in canines and was used to generate a vaccine for canine visceral leishmaniasis (Carrillo et al., 2008). Other proteins like unnamed proteins and hypothetical proteins, appeared in a high proportion in differential and immunogenic proteome (45%). Some of them could be valuable to develop vaccine, even though their characterization and function remain unknown. The 2-DE technique used in this study is the most classic proteomic analysis method. Currently, the most powerful 2D-gel based protein profiling technique is the 2D-difference gel electrophoresis (DIGE) technique (Lilley and Friedman, 2004), which has the advantage of being much more accurate and sensitive than the traditional 2-DE method. Additionally, a higher resolution LC-MS/MS technique could help identify other low abundance potential target proteins. MALDI methods could have missed out on these because of the fact that everything is being ionized at once. Future studies using these new technique may help us to identify more proteins as potential targets for vaccine development. In conclusion, 12 proteins were differentially expressed among the three developmental stages. A total of 16 proteins were recognized by rabbit and grouper anti-sera. Among these identified proteins, actin, ␣-tubulin, ␤-tubulin, enolase, and vacuolar ATP synthase catalytic subunit ␣ were considered as the most promising immunogenic proteins for vaccine development. Further studies will be conducted to evaluate the potential of the five selected antigens using the Tetrahymena expression system to express these proteins, and screen other candidate proteins using a large number of sera from naturally infected fish.

Acknowledgments This work was funded by the National Natural Science Foundation of China (Grant No. 31272681), Key projects of Fujian province for agriculture (2013NZ0002), the Science and Technology Program from Guangdong Province (Grant No. 2012A020800006), and the State Oceanic Administration Demonstration Projects for Innovation and Development of Marine Economy (GD2012-B01) to Pro. Anxing Li.

References Avilan, L., Gualdron-Lopez, M., Quinones, W., Gonzalez-Gonzalez, L., Hannaert, V., Michels, P.A., Concepcion, J.L., 2011. Enolase: a key player in the metabolism

and a probable virulence factor of trypanosomatid parasites-perspectives for its use as a therapeutic target. Enzyme Res. 2011, 932549. Bai, J.S., Xie, M.Q., Zhu, X.Q., Dan, X.M., Li, A.X., 2008. Comparative studies on the immunogenicity of theronts, tomonts and trophonts of Cryptocaryon irritans in grouper. Parasitol. Res. 102, 307–313. Barenco, P.V., Lourenco, E.V., Cunha-Junior, J.P., Almeida, K.C., Roque-Barreira, M.C., Silva, D.A., Araujo, E.C., Coutinho, L.B., Oliveira, M.C., Mineo, T.W., Mineo, J.R., Silva, N.M., 2014. Toxoplasma gondii 70 kDa heat shock protein: systemic detection is associated with the death of the parasites by the immune response and its increased expression in the brain is associated with parasite replication Toxoplasma gondii 70 kDa heat shock protein: systemic detection is associated with the death of the parasites by the immune response and its increased expression in the brain is associated with parasite replication. PLoS One 9, e96527. Burgess, P.J., Matthews, R.A., 1994. A standardized method for the in vivo maintenance of Cryptocaryon irritans (Ciliophora) using the grey mullet Chelon labrosus as an experimental host. J. Parasitol. 80, 288–292. Campos, T.D., Young, N.D., Korhonen, P.K., Hall, R.S., Mangiola, S., Lonie, A., Gasser, R.B., 2014. Identification of G protein-coupled receptors in Schistosoma haematobium and S. mansoni by comparative genomics. Parasit. Vectors 7, 242. Cardeilhac, P.T., Whitaker, B.R., 1988. Tropical fish medicine: copper treatments. Uses and precautions. Vet. Clin. North Am. Small Anim. Pract. 18, 435–448. Carrillo, E., Crusat, M., Nieto, J., Chicharro, C., Thomas Mdel, C., Martinez, E., Valladares, B., Canavate, C., Requena, J.M., Lopez, M.C., Alvar, J., Moreno, J., 2008. Immunogenicity of HSP-70, KMP-11 and PFR-2 leishmanial antigens in the experimental model of canine visceral leishmaniasis. Vaccine 26, 1902–1911. Chen, N., Xu, M.J., Nisbet, A.J., Huang, C.Q., Lin, R.Q., Yuan, Z.G., Song, H.Q., Zhu, X.Q., 2011. Ascaris suum: RNAi mediated silencing of enolase gene expression in infective larvae Ascaris suum: RNAi mediated silencing of enolase gene expression in infective larvae. Exp. Parasitol. 127, 142–146. Chen, N., Yuan, Z.G., Xu, M.J., Zhou, D.H., Zhang, X.X., Zhang, Y.Z., Wang, X.W., Yan, C., Lin, R.Q., Zhu, X.Q., 2012. Ascaris suum enolase is a potential vaccine candidate against ascariasis Ascaris suum enolase is a potential vaccine candidate against ascariasis. Vaccine 30, 3478–3482. Cheng, C.A., John, J.A., Wu, M.S., Lee, C.Y., Lin, C.H., Lin, C.H., Chang, C.Y., 2006. Characterization of serum immunoglobulin M of grouper and cDNA cloning of its heavy chain. Vet. Immunol. Immunopathol. 109, 255–265. Clark, T.G., Gao, Y., Gaertig, J., Wang, X., Cheng, G., 2001. The I-antigens of Ichthyophthirius multifiliis are GPI-anchored proteins. J. Eukaryot. Microbiol. 48, 332–337. Colorni, A., Burgess, P., 1997. Cryptocaryon irritans Brown 1951, the cause of ‘white spot disease’ in marine fish an update Cryptocaryon irritans Brown 1951, the cause of ‘white spot disease’ in marine fish an update. Aquar. Sci. Conserv. 1, 217–238. Dan, X.M., Li, A.X., Lin, X.T., Teng, N., Zhu, X.Q., 2006. A standardized method to propagate Cryptocaryon irritans on a susceptible host pompano Trachinotus ovatus. Aquaculture 258, 127–133. Ding, J., Bao, W., Liu, Q., Yu, Q., Abdille, M.H., Wei, Z., 2008. Immunoprotection of chickens against Eimeria acervulina by recombinant alpha-tubulin protein. Parasitol. Res. 103, 1133–1140. Ferreira, D., Cortez, M., Atayde, V.D., Yoshida, N., 2006. Actin cytoskeleton-dependent and -independent host cell invasion by Trypanosoma cruzi is mediated by distinct parasite surface molecules. Infect. immun. 74, 5522–5528. Gruber, G., Marshansky, V., 2008. New insights into structure–function relationships between archeal ATP synthase (A1A0) and vacuolar type ATPase (V1V0). Bioessays 30, 1096–1109. Hatanaka, A., Umeda, N., Hirazawa, N., 2008. Molecular cloning of a putative agglutination/immobilization antigen located on the surface of a novel agglutination/immobilization serotype of Cryptocaryon irritans. Parasitology 135, 1043–1052. Huang, X., Xu, Y., Guo, G., Lin, Q., Ye, Z., Yuan, L., Sun, Z., Ni, W., 2013. Molecular characterization of an actin depolymerizing factor from Cryptocaryon irritans. Parasitology 140, 561–568. Janke, C., Kneussel, M., 2010. Tubulin post-translational modifications: encoding functions on the neuronal microtubule cytoskeleton. Trends Neurosci. 33, 362–372. Kikuchi, S.A., Sodre, C.L., Kalume, D.E., Elias, C.G., Santos, A.L., de Nazare Soeiro, M., Meuser, M., Chapeaurouge, A., Perales, J., Fernandes, O., 2010. Proteomic analysis of two Trypanosoma cruzi zymodeme 3 strains. Exp. Parasitol. 126, 540–551. Kurup, S.P., Tewari, A.K., 2012. Induction of protective immune response in mice by a DNA vaccine encoding Trypanosoma evansi beta tubulin gene. Vet. Parasitol. 187, 9–16. Li, S.Q., Fung, M.C., Reid, S.A., Inoue, N., Lun, Z.R., 2007. Immunization with recombinant beta-tubulin from Trypanosoma evansi induced protection against T. evansi, T. equiperdum and T. brucei infection in mice. Parasite Immunol. 29, 191–199. Li, S.Q., Yang, W.B., Ma, L.J., Xi, S.M., Chen, Q.L., Song, X.W., Kang, J., Yang, L.Z., 2009. Immunization with recombinant actin from Trypanosoma evansi induces protective immunity against T. evansi, T. equiperdum and T. brucei infection. Parasitol. Res. 104, 429–435. Li, W., Su, Y.L., Mai, Y.Z., Li, Y.W., Mo, Z.Q., Li, A.X., 2014. Comparative proteome analysis of two Streptococcus agalactiae strains from cultured tilapia with different virulence. Vet. Microbiol. 170, 135–143.

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Libusova, L., Draber, P., 2006. Multiple tubulin forms in ciliated protozoan Tetrahymena and Paramecium species. Protoplasma 227, 65–76. Lilley, K.S., Friedman, D.B., 2004. All about DIGE: quantification technology for differential-display 2D-gel proteomics. Expert Rev. Proteomics 1, 401–409. Liu, Z.X., Liu, G.Y., Li, N., Xiao, F.S., Xie, H.X., Nie, P., 2012. Identification of immunogenic proteins of Flavobacterium columnare by two-dimensional electrophoresis immunoblotting with antibacterial sera from grass carp, Ctenopharyngodon idella (Valenciennes). J. Fish Dis. 35, 255–263. Luo, X.C., Xie, M.Q., Zhu, X.Q., Li, A.X., 2007. Protective immunity in grouper (Epinephelus coioides) following exposure to or injection with Cryptocaryon irritans. Fish Shellfish Immunol. 22, 427–432. Luo, X.C., Xie, M.Q., Zhu, X.Q., Li, A.X., 2008. Some characteristics of host-parasite relationship for Cryptocaryon irritans isolated from South China. Parasitol. Res. 102, 1269–1275. Lv, X., Huang, L., Chen, W., Wang, X., Huang, Y., Deng, C., Sun, J., Tian, Y., Mao, Q., Lei, H., Yu, X., 2014. Molecular characterization and serological reactivity of a vacuolar ATP synthase subunit epsilon-like protein from Clonorchis sinensis. Parasitol. Res. 113, 1545–1554. Matthews, B.F., Matthews, R.A., Burgess, P.J., 1993. Cryptocaryon irritans Brown, 1951 (Ichthyophthiriidae) the ultrastructure of the somatic cortex throughout the life cycle Cryptocaryon irritans Brown, 1951 (Ichthyophthiriidae) the ultrastructure of the somatic cortex throughout the life cycle. J. Fish Dis. 16 (4), 339–349. Misumi, I., Lewis, T.D., Takemura, A., Leong, J.A., 2011. Elicited cross-protection and specific antibodies in Mozambique tilapia (Oreochromis mossambicus) against two different immobilization serotypes of Cryptocaryon irritans isolated in Hawaii. Fish Shellfish Immunol. 30, 1152–1158. Nakanishi-Matsui, M., Sekiya, M., Nakamoto, R.K., Futai, M., 2010. The mechanism of rotating proton pumping ATPases. Biochim. Biophys. Acta 1797, 1343–1352. Porcel, B.M., Aslund, L., Pettersson, U., Andersson, B., 2000. Trypanosoma cruzi: a putative vacuolar ATP synthase subunit and a CAAX prenyl protease-encoding gene, as examples of gene identification in genome projects Trypanosoma cruzi: a putative vacuolar ATP synthase subunit and a CAAX prenyl protease-encoding gene, as examples of gene identification in genome projects. Exp. Parasitol. 95, 176–186. Priya, T.A.J., Lin, Y.H., Wang, Y.C., Yang, C.S., Chang, P.S., Song, Y.L., 2012. Codon changed immobilization antigen (iAg), a potent DNA vaccine in fish against Cryptocaryon irritans infection. Vaccine 30, 893–903. Rigos, G., Karagouni, E., Kyriazis, I., Athanasiou, E., Grigorakis, K., Kotou, E., Katharios, P., 2013. In vitro and in vivo evaluation of quinine in gilthead sea bream, Sparus aurata naturally infected with the ciliate Cryptocaryon irritans. Aquaculture 416, 185–191. Schappi, J.M., Krbanjevic, A., Rasenick, M.M., 2014. Tubulin, actin and heterotrimeric G proteins: coordination of signaling and structure. Biochim. Biophys. Acta 1838, 674–681.

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Skillman, K.M., Daher, W., Ma, C.I., Soldati-Favre, D., Sibley, L.D., 2012. Toxoplasma gondii profilin acts primarily to sequester G-actin while formins efficiently nucleate actin filament formation in vitro. Biochemistry 51, 2486–2495. Sun, H.Y., Zhu, X.Q., Xie, M.Q., Wu, X.Y., Li, A.X., Lin, R.Q., Song, H.Q., 2006. Characterization of Cryptocaryon irritans isolates from marine fishes in Mainland China by ITS ribosomal DNA sequences. Parasitol. Res. 99, 160–166. Wang, L., Cui, J., Hu, D.D., Liu, R.D., Wang, Z.Q., 2014. Identification of early diagnostic antigens from major excretory-secretory proteins of Trichinella spiralis muscle larvae using immunoproteomics. Parasit. Vectors 7, 40. Wang, X., Chen, W., Hu, F., Deng, C., Zhou, C., Lv, X., Fan, Y., Men, J., Huang, Y., Sun, J., Hu, D., Chen, J., Yang, Y., Liang, C., Zheng, H., Hu, X., Xu, J., Wu, Z., Yu, X., 2011. Clonorchis sinensis enolase: identification and biochemical characterization of a glycolytic enzyme from excretory/secretory products. Mol. Biochem. Parasitol. 177, 135–142. Wilson, A.P., Thelen, J.J., Lakritz, J., Brown, C.R., Marsh, A.E., 2004. The identification of a sequence related to apicomplexan enolase from Sarcocystis neurona. Parasitol. Res. 94, 354–360. Xu, D.H., Klesius, P.H., Shoemaker, C.A., 2009. Effect of immunization of channel catfish with inactivated trophonts on serum and cutaneous antibody titers and survival against Ichthyophthirius multifiliis. Fish Shellfish Immunol. 26 (4), 614–618. Xu, H.J., Lin, J.G., Zhang, C.X., Suzuki, S., 2008. Expression and immunogenic comparison of VP2 and VP3 from marine birnavirus. J. Fish Dis. 31, 297–304. Yambot, A.V., Song, Y.L., 2006. Immunization of grouper, Epinephelus coioides, confers protection against a protozoan parasite, Cryptocaryon irritans. Aquaculture 260, 1–9. Yang, J., Qiu, C., Xia, Y., Yao, L., Fu, Z., Yuan, C., Feng, X., Lin, J., 2010. Molecular cloning and functional characterization of Schistosoma japonicum enolase which is highly expressed at the schistosomulum stage. Parasitol. Res. 107, 667–677. Yin, L.T., Hao, H.X., Wang, H.L., Zhang, J.H., Meng, X.L., Yin, G.R., 2013. Intranasal immunisation with recombinant Toxoplasma gondii actin partly protects mice against toxoplasmosis. PLoS One 8, e82765. Yoshinaga, T., 2001. Effects of high temperature and dissolved oxygen concentration on the development of Cryptocaryon irritans (Ciliophora) with a comment on the Autumn outbreaks of cryptocaryoniasis. Fish Pathol. 36, 231–235. Zhou, D.H., Zhao, F.R., Nisbet, A.J., Xu, M.J., Song, H.Q., Lin, R.Q., Huang, S.Y., Zhu, X.Q., 2014. Comparative proteomic analysis of different Toxoplasma gondii genotypes by two-dimensional fluorescence difference gel electrophoresis combined with mass spectrometry. Electrophoresis 35, 533–545.

Please cite this article in press as: Mai, Y.-Z., et al., Proteomic analysis of differentially expressed proteins in the marine fish parasitic ciliate Cryptocaryon irritans. Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2015.05.004