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Journal of Aquatic Animal Health Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/uahh20

Comparative Analysis of the Acute Response of Zebrafish Danio rerio Skin to Two Different Bacterial Infections a

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a

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Aijun Lü , Xiucai Hu , Yi Wang , Xiaojing Shen , Aihua Zhu , Lulu Shen , Qinglei Ming a

& Zhaojun Feng

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School of Life Sciences , Jiangsu Normal University , Xuzhou , 221116 , China

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Key Laboratory for Biotechnology on Medicinal Plants of Jiangsu Province , Xuzhou , 221116 , China Published online: 15 Nov 2013.

To cite this article: Aijun Lü , Xiucai Hu , Yi Wang , Xiaojing Shen , Aihua Zhu , Lulu Shen , Qinglei Ming & Zhaojun Feng (2013) Comparative Analysis of the Acute Response of Zebrafish Danio rerio Skin to Two Different Bacterial Infections, Journal of Aquatic Animal Health, 25:4, 243-251, DOI: 10.1080/08997659.2013.829132 To link to this article: http://dx.doi.org/10.1080/08997659.2013.829132

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Journal of Aquatic Animal Health 25:243–251, 2013  C American Fisheries Society 2013 ISSN: 0899-7659 print / 1548-8667 online DOI: 10.1080/08997659.2013.829132

ARTICLE

Comparative Analysis of the Acute Response of Zebrafish Danio rerio Skin to Two Different Bacterial Infections ¨ Aijun Lu* School of Life Sciences, Jiangsu Normal University, Xuzhou 221116, China

Xiucai Hu

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Key Laboratory for Biotechnology on Medicinal Plants of Jiangsu Province, Xuzhou 221116, China

Yi Wang, Xiaojing Shen, Aihua Zhu, Lulu Shen, Qinglei Ming, and Zhaojun Feng School of Life Sciences, Jiangsu Normal University, Xuzhou 221116, China

Abstract Skin is an important innate immune organ in fish; however, little is known about the skin’s immune response to infectious pathogens. We conducted a comparative analysis of the acute immune response of Zebrafish Danio rerio skin against gram-positive (Staphylococcus chromogenes) and gram-negative (Citrobacter freundii) bacterial infections. Gene expression profiles induced from the two different infections were identified by microarray hybridization, with many genes demonstrating an acute immune response in the skin. Differentially expressed genes were mainly involved in response to stress and stimulus, complement activation, acute-phase response, and defense and immune response. Compared with transcription patterns of skin from the two infections, a similar innate immunity (e.g., transferrin, coagulation factor, complements, and lectins) was observed but with different acute-phase genes (e.g., ceruloplasmin, alpha-1-microglobulin, vitellogenin, and heat shock protein). These results suggest that the skin of fish plays an important role in the innate immune responses to bacterial infection.

Skin is an essential protective barrier for fish and functions as a first line of defense against invading pathogens (Rakers et al. 2010). Although skin is an important component of the mucosal immune system, only a few studies have characterized the transcription profile of fish skin. One of the earliest investigations analyzed gene expression in the skin of Channel Catfish Ictalurus punctatus (Karsi et al. 2002). Subsequently, several studies reported regulation of immune-relevant gene expression in the skin of Rainbow Trout Oncorhynchus mykiss, Common Carp Cyprinus carpio, and Atlantic Salmon Salmo salar after ectoparasitic infections (Lindenstrøm et al. 2003; Gonzalez et al. 2007b; Skugor et al. 2008). Very recently, Li et al. (2013) investigated the transcriptional effects of Aeromonas hydrophila challenge in the skin of Channel Catfish by using an Agilent microarray and demonstrated that the A. hydrophila infection rapidly altered a

number of potentially critical lectins, chemokines, interleukins, and other mucosal factors in a manner that was predicted to enhance the pathogen’s ability to adhere to and invade the host. However, little is known about transcriptional profiles of the skin’s immune response to bacterial pathogens. As opportunistic pathogens, Citrobacter freundii and Staphylococcus chromogenes do not generally cause disease in healthy animal hosts (L¨u et al. 2011a, 2011b). However, opportunistic infections have become a serious health problem in intensive aquaculture in China (Chen et al. 2007). Citrobacter freundii is an aerobic, gram-negative bacillus that was first isolated from diseased fish in 1982 (Sato et al. 1982); it was subsequently isolated from Atlantic Salmon, Common Carp, catfish, and Rainbow Trout (Austin et al. 1992; Karunasagar et al. 1992; Jeremi´c et al. 2003; L¨u et al. 2011b). Staphylococcus chromogenes is a

*Corresponding author: [email protected] Received March 20, 2013; accepted July 19, 2013

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gram-positive, coagulase-negative staphylococcus that is a common component of normal skin flora and has been isolated from the skin of healthy swine (Devriese et al. 1978; Saito et al. 1996); it is recognized as the causative agent of exudative epidermitis in swine (Andresen et al. 2005). Additionally, S. chromogenes is a well-known pathogen associated with mastitis in cows (Devriese et al. 2002; Leitner et al. 2011) and has been reported as the cause of skin infection in goats (Andrews and Lamport 1997). Recently, Staphylococcus aureus and other Staphylococcus spp., including S. chromogenes, have been used in Zebrafish Danio rerio disease studies (Neely et al. 2002; Miller and Neely 2004; L¨u et al. 2011a). The Zebrafish has emerged as a valuable model for immunological studies (Miller and Neely 2004; Sullivan and Kim 2008). Recently, Zebrafish were used to evaluate the pathogenicity of C. freundii and S. chromogenes (L¨u et al. 2011a, 2011b), exhibiting clinical features of skin infection. In the present study, we conducted a comparative analysis of transcription profiles in the skin of Zebrafish infected with S. chromogenes and C. freundii, with the aim of understanding the immune responses of fish skin against gram-positive and gram-negative bacterial pathogens.

METHODS Pathogenicity assays in Zebrafish.—Wild-type Zebrafish adults (∼0.4 g in weight) were bought from a pet shop in Xuzhou, China. For 15 d prior to the experiment, Zebrafish were acclimatized in aerated freshwater tanks at a water temperature of 25◦ C and were given commercial dry feed (Fengnian) distributed by hand twice daily. The bacteria were cultured on modified nutrient agar plates at 28◦ C for 24 h, and the number of colony-forming units (CFU) was determined by the agar plate count method. Before S. chromogenes exposure, 60 fish were anesthetized in a 0.16-mg/mL solution of tricaine, and a sterile scalpel was use to remove several scales by scraping along the lateral surface behind the pectoral fins (Neely et al. 2002). After recovery from anesthesia, the scraped fish were exposed to S. chromogenes at 108 CFU/mL by immersion for 10 min. Control fish were treated the same except that they were not exposed to the pathogen. For C. freundii infection, the fish were challenged by immersion with 108 CFU/mL as previously described (L¨u et al. 2011b). Control fish were treated with physiological saline solution (0.65% NaCl) alone. The infected fish and control fish were held for 7 d after exposure to allow for observations of symptoms and mortality. At about 7 h postexposure, control and treatment fish were randomly collected, and the skin sections were aseptically excised from the dorsal regions along the two sides of the fish. The skin samples were pooled together in equal amounts, flash frozen in liquid nitrogen, and then stored at −80◦ C for total RNA extraction. RNA preparation, hybridization, and data analysis.—Total RNA was extracted from the skin by using Trizol reagent

(Invitrogen) and was further purified by using an RNeasy Mini Kit (Qiagen) according to the manufacturers’ instructions. Integrity of the RNA was determined by agarose gel electrophoresis and ethidium bromide staining. The RNA samples were hybridized separately with the same Affymetrix GeneChip probe arrays (containing 14,900 transcripts). Two replicates were performed in order to compare the gene expression profiles between the infected and the control samples. Methods for probe labeling and microarray hybridization were described previously (Wu et al. 2010). Briefly, 2 µg of total RNA were used to synthesize single- and double-stranded complementary DNA (cDNA); purified double-stranded cDNA was then converted into the biotin-labeled complementary RNA (cRNA) using MessageAmp II cRNA Amplification Kits (Ambion). Ten-microgram quantities of labeled cRNA were fragmented and hybridized to the Affymetrix Zebrafish Genome Array. Hybridization was performed at 45◦ C with rotation for 16 h on an Affymetrix GeneChip Hybridization Oven 640. The GeneChip arrays were washed and then stained (streptavidin– phycoerythrin) on an Affymetrix Fluidics Station 450, followed by scanning on an Affymetrix GeneChip Scanner 3000. The hybridization data were analyzed by using GeneChip Operating Software (GCOS) version 1.4. The scanned images were first assessed by visual inspection and then were analyzed to generate raw data files, which were saved as CEL files using the default setting of GCOS. A global scaling target intensity value of 500 was used to normalize the different arrays. The detection calls (present, absent, or marginal) for the probe sets were made by GCOS. The ratio of the expression values for infected fish and control fish was used to calculate x-fold changes. The differentially expressed genes were identified using Significant Analysis of Microarray (SAM) software (Tusher et al. 2001) and were selected on the basis of their x-fold changes (≥ 2.0-fold or ≤ 0.5-fold). Gene Ontology category and pathway analysis.—The categorization of biological process gene ontology (GO) was analyzed using the Gene Ontology project (www.geneontology. org). The pathway analysis was carried out using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Fisher’s test and the χ2 test were used to classify the GO category and pathway analysis, and the false discovery rate procedure was applied to correct the P-value (Benjamini and Yekutieli 2001). A P-value of 0.05 and a false discovery rate of 0.05 were used as thresholds for selecting significant GO categories and KEGG pathways. Quantitative real-time PCR.—Microarray data were validated by quantitative real-time PCR (qPCR), and five pairs of primers were designed by using the Primer 5 program (sequence not shown). Quantitative PCR was performed as described by Farmer et al. (2011). Briefly, qPCR involved amplifying cDNA with the SYBR Green qPCR Kit (Finnzymes) in a DNA Engine Opticon Continuous Fluorescence Detection System (MJ Research). Amplification conditions were 94◦ C for 4 min, followed by 40 cycles of 94◦ C for 20 s, 58◦ C for 20 s, and 72◦ C

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for 20 s. The expression of candidate genes was normalized by using β-actin as a housekeeping gene. Each gene was run in triplicate. After completion of the qPCR amplification, the relative x-fold change after stimulation was calculated based on the 2−CT method (Livak and Schmittgen 2001).

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RESULTS Infection of Zebrafish At approximately 12 h after exposure to S. chromogenes, obvious symptoms of discomfort in the Zebrafish were observed, including erratic swimming behavior, floating, or swimming abnormally near the water’s surface with an increased rate of respiration. All infected fish typically died after 24–72 h, exhibited hemorrhages on the skin, distended abdomens, perianal edema, and large lesions at and near the site of injury. Control fish were scraped but were not exposed to S. chromogenes and showed no signs of infection up to 7 d after treatment. Fish that were infected with C. freundii showed petechial hemorrhages on the gills and developed diffuse bleeding on the skin; the fish became lethargic, executed uncoordinated movements, and floated on the surface of the water. The infection kinetics were similar to those of S. chromogenes infection in that the fish died between 24 and 72 h, and obvious symptoms of discomfort were also observed at about 12 h after exposure. Comparative Analysis of the Gene Expression Profiles in Skin To identify immune-related genes that were induced by infection in the skin, the profiles of differential gene expression were obtained by using the microarray analysis (Table 1). In total, 175 genes were obtained from S. chromogenes-infected skin samples, and 229 genes were obtained from C. freundii-infected samples. Results showed that 88 genes from S. chromogenesinfected samples and 43 genes from C. freundii-infected samples shared similar annotations; these genes were mainly involved in complement activation (e.g., c1ql4, c3c, c3b, cfb, c8g, c8b, c7-1, and c9; gene abbreviations are defined in Table 1), acutephase response (e.g., tfa, cp, agt, a2ml, and ambpl), defense and immune response (e.g., ifn1, irf2bp1, lman1, lgals1l3, cxcr7b, btg4, mucin, ighmbp2l, tnfaip8, apoa1, ctsl1, and f5), and response to stress and stimulus (e.g., hsp90a1, hsp90a2, hsp70, sepp1b, mt2, vtg1-4, ucp4, and cyp; Table 2). Compared with transcription patterns of skin from the two bacterial infections, similar acute immune responses (e.g., transferrin, coagulation factor, complements, and lectins) were induced in the skin of

FIGURE 1. Validation of microarray-based gene expression profiles by quantitative real-time PCR (qPCR). The qPCR data were normalized to β-actin expression. Changes (x-fold) were determined by calculating the ratio of the mean expression values from the skin samples of control Zebrafish and fish that were experimentally infected with Citrobacter freundii (C.f) and Staphylococcus chromogenes (S.c). Five up-regulated genes were selected (tfa = transferrin; c3c = complement component c3c; vtg2 = vitellogenin 2; mbl-1 = mannosebinding lectin 1; f5 = coagulation factor V).

Zebrafish but with obviously different acute-phase genes (cp, ambpl, vtgs, hsps, etc.). In contrast, the skin was demonstrated to have some novel acute immune response genes in response to the microbial challenge, such as apoea, which had a 22-fold induction after the infection, and vtg1, which had a 118-fold induction. Furthermore, to confirm the data obtained from microarray analysis, five immune-related genes were selected for qPCR analysis. Results showed that the expression trends of the detected genes (i.e., tfa, c3c, vtg2, mbl-1, and f5) were generally consistent with the microarray data (Figure 1). The skin immune-related genes were classified into eight main functional categories according to GO annotation for biological process (Figure 2). The KEGG analyses of pooled data from S. chromogenes and C. freundii infections suggested that 22 and 49 pathways, respectively, may be involved in the acute immune responses of skin. Further pathway analysis showed that the skin’s acute immune response occurs via similar pathways involving cell cycle and communication, the mitogenactivated protein kinase signaling pathway, the tumor protein p53 signaling pathway, the wingless-type mouse mammary tumor virus integration site family (wnt) signaling pathway, and the calcium signaling pathway, among others. A comparison of the two global profiles against GO and KEGG analysis

TABLE 1. Global gene expression profiling in the skin of Zebrafish that were experimentally infected with Citrobacter freundii and Staphylococcus chromogenes (known/unknown = number of genes with known or unknown function).

Infection group

Up-regulated

Down-regulated

Known

Unknown

Total

C. freundii S. chromogenes

196 150

33 25

195 99

34 76

229 175

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TABLE 2. Differential expression of immune-related genes in Zebrafish skin screened by the complementary DNA microarray after the fish were experimentally infected with Citrobacter freundii and Staphylococcus chromogenes. Significantly up-regulated (≥ 2.0-fold change) and down-regulated (≤ 0.5-fold change) genes are shown.

Change (x-fold) Functional classification

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Complement activation

Acute-phase response

Response to stress and stimulus

Platelet activation and coagulation factors

Gene Complement component 8, beta polypeptide Complement component 8, gamma polypeptide Complement component 9 Complement components c3b/c3c Complement factor B Similar to complement component 1q, subcomponent-like 4 Similar to complement component c3b Similar to complement control protein factor I-B Similar to complement protein component C7-1 Alpha-1-microglobulin Alpha-2-macroglobulin-like Angiotensinogen Transferrin-a Ceruloplasmin Angiotensin I converting enzyme 2 Similar to alpha-2-macroglobulin precursor Heat shock cognate, 70-kilodalton protein Heat shock protein (HSP) 90, alpha 1 HSP 90, alpha 2 DnaJ (HSP 40) homolog subfamily B, member 1b Selenoprotein P plasma 1b Metallothionein 2 Vitellogenin 1 Vitellogenin 2 Vitellogenin 3, phosvitinless Vitellogenin 4 Uncoupling protein 4 Uncoupling protein 2 like Phenylalanine hydroxylase MUS81 endonuclease homolog Myeloid-specific peroxidase Matrix metalloproteinase 14 (membrane-inserted) alpha Mitogen-activated protein kinase interacting serine/threonine kinase 1 Cytochrome b5, type A (microsomal) Cytochrome P450, family 1, subfamily A Cytochrome P450, family 8, subfamily B, polypeptide 1 Cytochrome P450, family 3, subfamily A, polypeptide 65 Aquaporin 12 Apolipoprotein B like Aryl hydrocarbon receptor 2 Prostaglandin–endoperoxide synthase 2a Fibrinogen, alpha chain Fibrinogen, B beta polypeptide Fibrinogen, gamma polypeptide

Gene symbol

C. freundii

c8b c8g c9 c3b/c3c cfb c1ql4

2.288 2.158 3.975 2.478 4.448

LOC100149939 LOC557557 c7-1 ambpl a2ml agt tfa cp ace2 LOC562067 hsp70 hsp90a1 hsp90a2 dnajb1b sepp1b mt2 vtg1 vtg2 vtg3 vtg4 ucp4 ucp2l pah mus81 mpx mmp14a

2.286 0.343

S. chromogenes

2.644

2.081 2.578 15.063 3.847 6.36 2.238

3.001 2.699 10.895 0.458

6.855 2.685 2.637 2.328 5.929 2.249 118.699 14.575 0.482 85.812 4.079

2.061 2.264

2.547 2.431 5.328 2.366

mknk1

2.439

cyb5a cyp1a cyp8b1

2.541

cyp3a65

2.35

aqp12 apobl ahr2 ptgs2a fga fgb fgg

2.275 6.101 2.516 0.327 4.952 5.434 4.881

2.41 3.15 3.624

2.218

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ZEBRAFISH RESPONSE TO BACTERIAL INFECTIONS TABLE 2.

Continued.

Change (x-fold) Functional classification

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Defense and immune response

Gene Coagulation factor V Similar to coagulation factor VIII Thrombospondin 1 Interferon 1 Interferon regulatory factor 2 binding protein 1 Immunoglobulin mu binding protein 2 like Activating transcription factor 3 Apolipoprotein A-I Apolipoprotein A-IV Apolipoprotein Ea B-cell translocation gene 4 Calcium/calmodulin-dependent protein kinase II, delta 1 Cathepsin L1 CCAAT/enhancer binding protein (C/EBP) beta Cell division control protein 42, small effector 1 CC chemokine 1 Chemokine (C-X-C motif) receptor 7b Chemokine CCL-C11b Chymotrypsin-like Chymotrypsinogen B1 Collagen type X, alpha 1 Connexin 28-9 Death-associated protein 6 Early growth response 1 Elastase 2 Fatty acid-binding protein 2 Fatty acid-binding protein 6 Galactoside-binding lectin soluble 1 (galectin 1)-like 3 Growth arrest and DNA-damage-inducible alpha like Hexose-binding lectin 3 Insulin-like growth factor 2 messenger RNA binding protein 3 Inter-alpha (globulin) inhibitor H3 Mannose-binding lectin 1 5 -nucleotidase cytosolic II-like 1 Neutrophil cytosolic factor 1 Novel rhamnose-binding lectin ORAI calcium release-activated calcium modulator 1 Parvalbumin 9 Protein kinase interferon-inducible, double–stranded RNA-dependent inhibitor, repressor of (P58 repressor) Ras homolog gene family, member Gb Ras-like family 11, member B Similar to cell surface flocculin

Gene symbol f5 LOC100147976 thbs1 ifn1 irf2bp1 ighmbp2l atf3 apoa1 apoa4 apoea btg4 camk2d1 ctsl1 cebpb cdc42se1 ccl1 cxcr7b ccl-c11b ctrl ctrb1 col10a1 cx28-9 daxx egr1 ela2 fabp2 fabp6 lgals1l3

C. freundii 2.283 2.072 0.319 2.639 2.088 2.051 2.028 3.389

4.668 4.646 22.241 2.061 16.703

0.473 2.095 0.47 2.211 0.476 7.607 6.46 2.442 2.966 2.011 2.331 4.23 23.772 12.955 13.844 2.196 2.06 2.128

itih3 lman1 nt5c2l1 ncf1 CH211-250E5-11 orai1

0.488 2.3 4.411

pvalb9 prkrir

2.12

rhogb rasl11b LOC799913

2.027

2.972

gadd45al hbl3 igf2bp3

S. chromogenes

2.169 2.002 2.254

0.435

2.035 3.21 0.27 (Continued on next page)

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248 TABLE 2. Continued.

Change (x-fold)

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Functional classification

Apoptosis

Antigen processing and presentation

Cell adhesion and migration

Gene Similar to lipopolysaccharide-induced tumor necrosis factor (TNF) factor Similar to mucin 4 Similar to novel galactose-binding lectin domain-containing protein TNF receptor-associated factor (TRAF) and TNF receptor-associated protein Transmembrane channel-like 4 Trypsin TNF superfamily, member 10 like 4 TNF alpha-induced protein 8 Unc-51-like kinase 1 Villin 1 like Zebrafish Gene Collection (ZGC) 173545 B-cell lymphoma 2 (BCL2)-associated athanogene 4 BCL2-related ovarian killer b Caspase recruitment domain-like WD repeat domain 92 Programmed cell death 4b Programmed cell death 7 Tumor suppressor candidate 2a Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha a Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha b Major histocompatibility complex (MHC) class I, UAA gene MHC class I, UEA gene MHC class I, UFA gene Thy-1 cell surface antigen MHC class II, integral membrane protein, alpha chain 2 ZGC 92049 ZGC 64115 ZGC 64003 Scavenger receptor class B, member 1 Poliovirus receptor-related protein 3 One-eyed pinhead III-FBPL precursor wnt8-like protein 2/wingless-type mouse mammary tumor virus integration site family, member 8a Claudin c Claudin d Claudin f Claudin g Dead end

Gene symbol

C. freundii

LOC561592

0.481

LOC100000152 DKEYP-98A7-10

2.717 2.491

ttrap

2.177

tmc4 try tnfsf10l4 tnfaip8 ulk1 vil1l zgc:173545 bag4

S. chromogenes

2.393 5.16 0.473 2.172 2.116 5.284 2.81 2.216

bokb DKEY-1O2-6 wdr92 pdcd4b pdcd7 tusc2a nfkbiaa

2.956 3.564 3.697 2.288 2.144 0.41

nfkbiab

0.317

mhc1uaa

2.03

0.152

mhc1uea mhc1ufa thy1 mhc2a

3.427 2.81 2.064 0.415

zgc:92049 zgc:64115 zgc:64003 scarb1 pvrl3 oep LOC799904 wnt8-2/wnt8a

0.406 0.469 0.104 2.297 2.374 2.235 2.096 2.292

cldnc cldnd cldnf cldng dnd

3.206 2.459 2.412 3.545

3.645

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ZEBRAFISH RESPONSE TO BACTERIAL INFECTIONS

FIGURE 2. Gene Ontology categories of significant immune-related genes in the skin of Zebrafish that were experimentally infected with (a) Citrobacter freundii and (b) Staphylococcus chromogenes. [Figure available online in color.]

indicated that C. freundii and S. chromogenes infections shared 7 immune-related genes and 15 overlapping pathways.

DISCUSSION The Zebrafish is an ideal model organism for use in studies of the immune system (Miller and Neely 2004; Sullivan and Kim 2008). The skin of fish is considered the portal of entry as well as the site of attachment for most invading pathogens. Several studies have recently shown that skin is a peripheral immune organ in fish (Xu et al. 2002; Zhao et al. 2008). Despite the fact that skin provides the initial protection against pathogens, the amount of available information on the skin immune system and its responses is still very limited. The microarray has recently been used to identify host gene expression patterns in fish after bacterial infections such as Mycobacterium marinum, Edwardsiella ictaluri, Salmonella typhimurium, and Streptococcus suis (Meijer et al. 2005; Peatman et al. 2008; Elibol-Flemming et al. 2009; Stockhammer et al. 2009; Wu et al. 2010), but the previous studies did not focus on the skin’s immunity against bacteria.

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Skin Immune Responses Recent studies have shown that fish skin acts as a rich source of immune-related genes as well as an active modulator of local immune responses (Lindenstrøm et al. 2004; Gonzalez et al. 2007a, 2007b, 2007c). Most cytokines act in the local signaling network that is of considerable importance for the initiation, effectuation, and modulation of skin immune responses (Lindenstrøm et al. 2004; Caipang et al. 2011). Recently, a transcript analysis of skin was performed for Common Carp that were infected with the parasite Ichthyophthirius multifiliis (Gonzalez et al. 2007b), and the expression of several genes (e.g., complement components, interferon, and CC chemokine) was described, all of which may play regulatory roles in the skin immune system of fish. In addition, transcriptional profile analysis of skin from Atlantic Salmon and Atlantic Cod Gadus morhua showed high expression of genes involved in antibacterial activity, antiviral response, stress response, and anti-apoptotic activity (Caipang et al. 2011), as well as changes in the expression of genes associated with immune response, oxidative stress, protein folding, and cytoskeletal proteins (Skugor et al. 2008). In the present study, we report a greater number of immune-related genes than were previously reported for Zebrafish (Skugor et al. 2008; Caipang et al. 2011; Li et al. 2013), and they represent key genes for understanding the immune response of fish skin to infection. The increased expression of a number of different complement factors and acute-phase response proteins was indicative of an activated innate immune response in the skin of Zebrafish. A plausible explanation for the considerable number of immune-relevant genes observed in fish skin is the active role of the skin in implementing immune defense mechanisms against bacterial pathogens. It is worth noting that Zebrafish skin had some novel acute immune response genes to the bacterial pathogens, such as (1) vitellogenin 1 (vtg1), which exhibited a 118-fold induction after infection; and (2) apolipoprotein Ea (apoea), which exhibited a 22-fold induction. Recently, reports have shown that vitellogenin plays an important role in innate immune responses (Shi et al. 2006). Injection of lipopolysaccharide into Zebrafish induced a significant up-regulation of vitellogenin and inhibited growth of both Staphylococcus aureus and Escherichia coli (Tong et al. 2010). In addition to its part in lipid metabolism, the major apolipoprotein apoA-I has been proven to show antimicrobial activity in the skin of Common Carp (Concha et al. 2003) and also demonstrates antimicrobial activity in Rainbow Trout (Villarroel et al. 2007); apoA-I was recently classified as an acute-phase protein in fish (Audunsdottir et al. 2012). Conclusions In the present study, the acute immune responses of Zebrafish skin against bacterial infections with the gram-positive S. chromogenes and gram-negative C. freundii were compared. We observed some similarities in the gene expression patterns of representative gene sets, including complement activation, acute-phase response, response to stress and stimulus, and

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defense and immune response. Therefore, the similar results for the two different pathogens showed that fish skin might apply several common innate strategies to defend against those bacterial infections. Transcription profiles of skin have demonstrated obvious similarities between the response to bacteria and the response to ectoparasites (Gonzalez et al. 2007b; Skugor et al. 2008; Tadiso et al. 2011). The similar patterns of innate immunity and induction of complement components implied that a highly conserved mechanism is adopted by fish skin. The discovery of apolipoprotein and vitellogenin as the major acute immune response proteins may provide a novel mechanism for innate immunity of skin. In addition, our results demonstrated some significant signaling cascades, including the mitogen-activated protein kinase, wnt, and p53 signaling pathways in Zebrafish skin after infections with S. chromogenes and C. freundii. However, further studies are needed to determine the expression of genes in fish skin as well as their roles in the immune response against bacteria. ACKNOWLEDGMENTS This study was supported by the National Natural Science Foundation of China (Number 31272692 and Number 30800847), the Jiangsu Government Scholarship for Overseas Studies (2009), and the Priority Academic Program Development of Jiangsu Higher Education Institutions. REFERENCES Andresen, L. O., P. Ahrens, L. Daugaard, and V. Bille-Hansen. 2005. Exudative epidermitis in pigs caused by toxigenic Staphylococcus chromogenes. Veterinary Microbiology 105:291–300. Andrews, A. H., and A. Lamport. 1997. Isolation of Staphylococcus chromogenes from an unusual case of impetigo in a goat. Veterinary Record 140:584. Audunsdottir, S. S., B. Magnadottir, B. Gisladottir, Z. O. Jonsson, and B. T. Bragason. 2012. The acute phase response of Cod (Gadus morhua L.): expression of immune response genes. Fish and Shellfish Immunology 32:360–367. Austin, B., M. Stobie, and P. A. W. Robertson. 1992. Citrobacter freundii: the cause of gastroenteritis leading to progressive low level mortalities in farmed Rainbow Trout, Oncorhynchus mykiss Walbaum, in Scotland. Bulletin of the European Association of Fish Pathologists 12:166–167. Benjamini, Y., and D. Yekutieli. 2001. The control of the false discovery rate in multiple testing under dependency. Annals of Statistics 29:1165–1188. Caipang, C. M., C. C. Lazado, M. F. Brinchmann, J. H. Rombout, and V. Kiron. 2011. Differential expression of immune and stress genes in the skin of Atlantic Cod (Gadus morhua). Comparative Biochemistry and Physiology Part D Genomics Proteomics 6:158–162. Chen, J. X., C. T. Guang, H. Xu, Z. X. Chen, P. Xu, X. M. Yan, Y. T. Wang, and J. F. Liu. 2007. A review of cage and pen aquaculture: China. FAO (Food and Agriculture Organization of the United Nations). Fisheries Technical Paper 498:53–66. Concha, M. I., S. Molina, C. Oyarzun, J. Villanueva, and R. Amthauer. 2003. Local expression of apolipoprotein A-I gene and a possible role for HDL in primary defence in the carp skin. Fish and Shellfish Immunology 14:259–273. Devriese, L. A., M. Baele, M. Vaneechoutte, A. Martel, and F. Haesebrouk. 2002. Identification and antimicrobial susceptibility of Staphylococcus chromogenes isolates from intramammary infections of dairy cows. Veterinary Microbiology 87:175–182. Devriese, L. A., V. Hajek, P. Oeding, S. A. Meyer, and K. H. Schleifer. 1978. Staphylococcus hyicus (Sompolinsky, 1953) comb. nov. and Staphylococcus

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Comparative analysis of the acute response of zebrafish Danio rerio skin to two different bacterial infections.

Skin is an important innate immune organ in fish; however, little is known about the skin's immune response to infectious pathogens. We conducted a co...
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