Molecular characterization of polymeric immunoglobulin receptor and expression response to Aeromonas hydrophila challenge in Carassius auratus

Accepted Manuscript Molecular characterization of polymeric immunoglobulin receptor and expression response to Aeromonas hydrophila challenge in Carassius auratus Li Wang, Jie Zhang, Xianghui Kong, Chao Pei, Xianliang Zhao, Li Li PII:

S1050-4648(17)30550-8

DOI:

10.1016/j.fsi.2017.09.031

Reference:

YFSIM 4821

To appear in:

Fish and Shellfish Immunology

Received Date: 30 March 2017 Revised Date:

4 September 2017

Accepted Date: 9 September 2017

Please cite this article as: Wang L, Zhang J, Kong X, Pei C, Zhao X, Li L, Molecular characterization of polymeric immunoglobulin receptor and expression response to Aeromonas hydrophila challenge in Carassius auratus, Fish and Shellfish Immunology (2017), doi: 10.1016/j.fsi.2017.09.031. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Molecular characterization of polymeric immunoglobulin receptor and

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expression response to Aeromonas hydrophila challenge in Carassius auratus

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Li Wang1,2, Jie Zhang2, Xianghui Kong1,2,*, Chao Pei2, Xianliang Zhao2, Li Li2

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1 College of Life Science, Henan Normal University, Henan province, PR China

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2 College of Fisheries, Henan Normal University, Henan province, PR China

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*Correspondence: Xianghui Kong

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E-mail: [email protected];

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Telephone Number: 86-373-3328507;

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Postal Address: No. 46, Jianshe Road, College of Fisheries, Henan Normal University, Xinxiang

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453007, PR China

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ACCEPTED MANUSCRIPT Abstract

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The polymeric immunoglobulin receptor (pIgR) plays a pivotal role in mucosal immune response by

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transporting polymeric immunoglobulins onto the surface of mucosal epithelia to protect animals

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from invading pathogens. In this study, the full-length cDNA of pIgR was firstly cloned in Qihe

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crucian carp (Carassius auratus), hereafter designated as CapIgR, by using reverse transcription

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polymerase chain reaction and rapid amplification of cDNA ends. The molecular characterization

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and expression of CapIgR were investigated. The full-length cDNA sequence of CapIgR was

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composed of 1409 bp, which included a 112 bp 5ʹ-untranslated region (UTR), a 984 bp ORF, and a

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313 bp 3ʹ-UTR, with a putative polyadenylation signal sequence AATAAA located upstream of the

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poly(A) tail. The deduced amino acid sequence indicated that CapIgR was a single-spanning

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transmembrane protein with 327 amino acids and possessed a signal peptide, an extracellular region

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containing two immunoglobulin-like domains, a transmembrane region, and an intracellular region.

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The mRNA expression levels of CapIgR were detected in different tissues of healthy C. auratus by

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quantitative real-time PCR, and the highest expression level was found in the liver. After Aeromonas

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hydrophila challenge, CapIgR expression was upregulated in different tissues at certain time points,

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and temporal expression changes of CapIgR fluctuated in a time-dependent manner. CapIgR

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exhibited rapid immune response to A. hydrophila challenge and played an important role in the

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immune defense of fish. These findings provided insights into the structure, function, and immune

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defense mechanism of CapIgR in C. auratus. This study can serve as a basis for developing disease

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control strategies in aquaculture.

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Key word: Polymeric immunoglobulin receptor; Qihe crucian carp Carassius auratus; Aeromonas

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hydrophila; Immune defense.

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1. Introduction As the first line of specific immune defense, the mucosal immune system protects organisms

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from a wide range of pathogens. In the mammalian mucosa, polymeric immunoglobulin receptor

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(pIgR), a key component of immune defense, mediates polymeric immunoglobulin (dimeric IgA and

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in some mammals, pentameric IgM) transcytosis from the basolateral surface to the apical surface of

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epithelial cells [1-3], followed by the release of secretory IgA (sIgA) into mucosal secretions. The

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polymeric IgA transcytosis via pIgR ensures that sIgA functions as an immunological barrier by

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blocking the adherence and invasion of pathogens and promotes the intracellular neutralization of

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pathogenic microorganisms [2,4,5]. The pIgR cDNA of chicken (Gallus gallus) has been cloned [6],

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whereas the pIgR of African clawed frog (Xenopus laevis) has been characterized [7]. These studies

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have indicated that pIgR plays an important role in immune defense in mammals, avians, and

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amphibians.

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pIgR has been characterized in various fish species, including fugu (Takifugu rubripes) [3],

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common carp (Cyprinoid carpio) [8], orange-spotted grouper (Epinephelus coioides) [9], rainbow

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trout (Oncorhynchus mykiss) [10], Atlantic salmon (Salmo salar) [11], olive flounder (Paralichthys

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olivaceus) [12], turbot (Scophthalmus maximus) [13], zebrafish (Danio rerio) [14], Atlantic cod

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(Gadus morhua) [15], and sea bass (Lateolabrax japonicus) [16]. The pIgR is highly expressed in the

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liver, intestine, skin, and gill of fish, indicating similar tissue specificity in fugu, common carp,

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grouper, salmon, flounder, zebrafish, and sea bass [3,8,9,11,12,14,16]. The findings implied that fish

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pIgR, similar to mammalian pIgR, could be involved in mucosal defense. Moreover, pIgR expression

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in fish has been induced by Gram-negative bacteria (Vibrio anguillarum) [17], Gram-positive

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bacteria (Streptococcus iniae), and viruses (Snakehead rhabdovirusm, SHRV) [14]. In the common

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carp, the mRNA expression of pIgR in the hindgut after V. anguillarum immersion initially increases

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but subsequently decreases [17], the pattern of which is similar to pIgR expression in the skin, gill,

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stomach, intestine, head-kidney, and spleen of turbot [13]. In addition, the mRNA expression level of

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zebrafish pIgR is upregulated after S. iniae infection but decreases after SHRV infection [14].

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Notably, Yang et al. [16] found that pIgR in sea bass could interact with Escherichia coli, Aeromonas

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hydrophila, Staphylococcus aureus, and Bacillus subtilis, and speculated that pIgR could function in

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the recognition of invading bacteria. As evident, only few reports focused on the immune response of

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pIgR to pathogenic bacteria in fish, including common carp, turbot, and zebrafish. Hence, the

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mechanism underlying the immune response of fish pIgR remains unclear. The Qihe crucian carp Carassius auratus is a gynogenetic triploid freshwater fish that naturally

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lives in Qihe River. It is famous for its rich nutrition and delicious taste and is widely cultured in the

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north of Henan Province, China [18]. However, the rapid decline in C. auratus due to environmental

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pollution, overfishing, and pathogenic microorganisms has caused extensive economic losses in the

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aquaculture in the past decades. A. hydrophila, a common Gram-negative bacterium, infects fish and

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other animals and causes bacterial septicemia [19]. This devastating acute infectious disease spreads

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quickly and causes extensive economic losses in freshwater-cultured Cyprinid fish, including C.

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carassius, silver carp (Hypophthalmichthys molitrix), and bluntnose black bream (Megalobrama

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amblycephala) [20-23].

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In consideration of the pivotal role of pIgR in the immune defense of fish, characterization of

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the sequence and structure of pIgR and elucidation of its role in the immune response to bacteria are

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important. To date, only few studies have focused on C. auratus pIgR. Thus, the present study

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cloned the pIgR gene in C. auratus and determined its immune response to A. hydrophila. This study

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aimed to characterize pIgR sequence and structure and to elucidate the immune response of C.

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auratus to A. hydrophila. This study will contribute to the understanding of immune defense against

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infectious diseases in C. auratus and help develop techniques to control disease outbreaks in

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aquaculture.

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2. Materials and methods

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2.1. Experimental fish and tissues sampling

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Healthy C. auratus (35 ± 5 g) were obtained from the breeding farm of Qihe crucian carp in

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Henan Province. The fish were randomly grouped and cultured in aerated freshwater at 20 ± 2 °C in

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plastic aquaria and fed with commercial pellets twice a day. The experiments were performed in

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triplicate. Prior to experimentation, the fish were acclimatized for 2 weeks. Healthy fish tissues,

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including gill, liver, spleen, kidney, head-kidney, intestine, skin, and muscle, were sampled for

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detecting the mRNA expression profile of CapIgR.

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2.2. Bacteria

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The pathogen A. hydrophila was separated from C. auratus in our laboratory. Prior to the

ACCEPTED MANUSCRIPT challenge, A. hydrophila was cultured in Luria–Bertani medium at 28 °C for 12 h with constant

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shaking (200 rpm). The bacteria were harvested by centrifugation at 6000 rpm for 10 min, washed

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three times with 0.65% NaCl, and finally resuspended in 0.65% NaCl. The concentration of the

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bacteria was determined as colony forming unit (CFU) by plating following 10-fold serial dilutions

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on agar plates.

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2.3. Bacterial challenge experiment

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To study the expression response of CapIgR to A. hydrophila challenge, the fish were randomly

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divided into two groups (30 fish per group). In the experimental group, each fish was

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intraperitoneally injected with 100 µL of 4 × 106 CFU/mL A. hydrophila suspended in 0.65% NaCl.

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The experimental concentration of the bacterial suspension was designed based on the median lethal

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concentration (LC50) of 1.1 × 107 CFU/mL in the preliminary experiments. In the control group, each

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fish was intraperitoneally injected with 100 µL of 0.65% NaCl. After A. hydrophila injection, the

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liver, spleen, head-kidney, intestine, skin, and gill were collected at 3, 6, 12, 24, and 48 h. The

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samples were immediately preserved in liquid nitrogen and then stored at −80 °C until RNA

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extraction. The experiments were performed in triplicate.

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2.4. Total RNA extraction and first-strand cDNA synthesis

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Total RNA was isolated from the liver of C. auratus using TRIzol Reagent (TaKaRa, Japan) in

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accordance with the manufacturer’s protocol. RNA concentrations were measured using a NanoDrop

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2000 spectrophotometer (Thermo Scientific, USA), and the integrity of RNA was assessed by

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electrophoresis on 1.5% agarose gel. First-strand cDNA synthesis was carried out using the

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PrimeScriptTM II 1st Strand cDNA Synthesis Kit (TaKaRa, Japan) to clone the full-length cDNA

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sequence of pIgR. The cDNA templates were stored at −20 °C until use.

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To evaluate the mRNA expression of pIgR in various tissues of fish, total RNA of each sample

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was extracted as described above, and then first-strand cDNA was synthesized using PrimeScript ™

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RT Master Mix (Perfect Real Time) (Takara, Japan) with 500 ng of total RNA.

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2.5. Molecular cloning of the full-length cDNA sequence of CapIgR

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To clone the central cDNA sequence of CapIgR, degenerate primers (Table 1) were designed

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using software Primer 5.0 based on the conserved pIgR cDNA sequences from other fish. PCR was

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carried out in accordance with the manufacturer’s protocol in a 20 µL reaction system consisting of 1

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µL of cDNA template, 1 µL of each primer (10 mM), 10 µL of 2× Taq Premix™ (TaKaRa, Japan),

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and 7 µL of ddH2O. The cycle profile of PCR was as follows: 95 °C for 5 min, 35 cycles (95 °C for

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30 s, 58 °C for 1 min, and 72 °C for 30 s), and 72 °C for 3 min. The PCR products were cloned into

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the pMD19-T vector (TaKaRa, Japan), and then transformed into E. coli DH5α (Biomed, China).

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Positive clones were sequenced in Sangon Biotech Company (Shanghai, China). Two pairs of specific primers (pIgR-3ʹ-Out and pIgR-3ʹ-In, pIgR-5ʹ-Out and pIgR-5ʹ-In) were

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designed based on the central cDNA sequence of CapIgR. The full-length cDNA sequence of CapIgR

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was obtained using the rapid amplification of cDNA ends (RACE) method as previously described

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[24]. The second PCR products of 3ʹ RACE and 5ʹ RACE were cloned and sequenced as described

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above. The full-length cDNA sequence of CapIgR was obtained through assembly of overlapping

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sequences. On the basis of the assembled cDNA sequence, the specific primers (pIgR-F and pIgR-R)

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(Table 1) were designed to confirm the accuracy of the full length of CapIgR.

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2.6. Bioinformatics analysis

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Homologous analysis of pIgR cDNA sequences and the deduced amino acid sequences was

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performed using the BLAST program of NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The open

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reading frame (ORF) was identified using EditSeq in DNAStar. The predicted molecular weight (Mw)

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and isoelectronic point (pI) were determined using Compute pI/Mw (http://web.expasy.org/compute

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pi/). Domain prediction and annotation were conducted using the Simple Modular Architecture

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Research Tool (SMART, http://smart.emblheidelberg.de/). Signal peptide and transmembrane region

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of the protein were predicted using SignalIP 4.1 (http://www.cbs.dtu.dk/services/SignalP/) and

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TMHMM2.0 (http://www.cbs.dtu.dk/services/TMHMM/), respectively. Glycosylation of the protein

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was analyzed using NetNGlyc 1.0 Server (http://www.cbs.dtu.dk/services/NetNGlyc/) and NetOGlyc

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4.0 Server (http://www.cbs.dtu.dk/services). Multiple alignments of the amino acid sequences were

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performed using Clustal X (http://www.clustal.org/clustal2/). On the basis of the amino acid

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sequences of the pIgR from vertebrates, a phylogenetic tree was constructed using the

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neighbor-joining method in MEGA 7.0 with a bootstrap repetition number of 2000.

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2.7. The mRNA expression of CapIgR in C. auratus

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The mRNA expression levels of CapIgR in the different tissues and its temporal mRNA

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expression changes after A. hydrophila infection were determined using the AceQ® qPCR SYBR®

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Green Master Mix (Vazyme Biotech, China) on the Applied Biosystems (ABI) 7500 Real Time PCR

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System (ABI, USA) in accordance with the manufacturer’s protocol. The primers of qpIgR-F and

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qpIgR-R (Table 1) were used to amplify a 236 bp fragment of pIgR. The primers of β-actin-F and

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β-actin-R (Table 1) were used to amplify the internal control gene β-actin. The quantitative real-time PCR (qRT-PCR) amplification for each sample was performed in a

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20 µL reaction volume consisting of 2 µL of the cDNA template, 10 µL of 2×AceQ® qPCR SYBR®

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Green Master Mix, 0.4 µL of each primer (10 µM), and 7.2 µL of ddH2O. The cycle profile was

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performed as follows: 95 °C for 5 min, followed by 40 cycles at 95 °C for 10 s and at 60 °C for 30 s.

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Each sample was examined in triplicate. The relative mRNA expression of pIgR was calculated using

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the comparative threshold cycle method (2-∆∆Ct) [25]. All data were processed using SPSS 19.0, and

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the significant difference between the control and challenged groups at each time point was assessed

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by one-way ANOVA. Significant difference was set at P < 0.05, and extremely significant difference

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was set at P < 0.01.

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Table 1 Primers used in this study.

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Primer

Sequence (5ʹ-3ʹ)

CCCACATCTGGACATAGTTTARG

pIgR-R1

ACGACTGTRAGRKTCAKTTGAG

3ʹ RACE Olig(T)-Adaptor

CTGATCTAGAGGTACCGGATCC(T)14

3ʹ RACE Adaptor

CTGATCTAGAGGTACCGGATCC

pIgR-3 ʹ-Out

GCATGGTGACGGACTCTGAAGG

pIgR-3 ʹ-In

pIgR-F pIgR-R

GACTCGAGTCGACATCG TCACTGGACCTGAGGCTTTTACT

central

3ʹ RACE

5ʹ RACE

TCCACTCCCGTTAGGGGCTGATT

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pIgR-5 ʹ-In

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GACTCGAGTCGACATCGA(T)17

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pIgR-5 ʹ-Out

Amplify sequence

GGAGCTGGTCAACAGAAGGTGG

5ʹ RACE Olig(T)-Adaptor 5ʹ RACE Adaptor

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pIgR-F1

Application

CCCACATCTGGACATAGTTTAGG

TATAATAAAATATTACTCCAGT

qpIgR-F

GGTGACGGACTCTGAAGGAA

qpIgR-R

GCCTGTGAGGGTCATTTGAG

β-actin-F

CATTGACTCAGGATGCGGAAACT

β-actin-R

CTGTGAGGGCAGAGTGGTAGACG

Confirm the full length cDNA

qRT-PCR

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3. Results

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3.1. cDNA cloning and molecular characterization of CapIgR

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The full-length cDNA of CapIgR was obtained by RT-PCR and RACE, and it was submitted to

ACCEPTED MANUSCRIPT the GenBank database (Accession No. KY652915). The complete cDNA sequence of CapIgR was

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1409 bp, which included a 112 bp 5ʹ untranslated region (UTR), a 984 bp ORF, and a 313 bp 3ʹ UTR,

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with a putative polyadenylation signal sequence AATAAA located upstream of the poly(A) tail (Fig.

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1). Analysis of the deduced amino acid sequence of CapIgR using SMART indicated that CapIgR

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was a single-spanning transmembrane protein with 327 amino acids and comprised a signal peptide,

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an extracellular region, a transmembrane region, and an intracellular region. The extracellular region

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of CapIgR contained two Ig-like domains (ILDs), which was in accordance with the results reported

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in other fish (Fig. 2). ILD1 (25–128) and ILD2 (137–229) were composed of 104 and 93 amino acids,

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respectively, linked by a peptide of eight amino acids (Figs. 1 and 2). The Mw and pI of CapIgR

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were 36.4 kDa and 6.17, respectively. Ten O-glycosylation residues and only one N-glycosylation

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residue were predicted in the extracellular region.

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CCCACATCTGGACATAGTTTAGGGCAGAAGGAAAGTGGGAGTGAATGCAGAAATATTCCTTCTCACATCAGCTGTCTTTAGTAAGAGGGC CTTTTCTCACTCCACCTCAGCGatgactcttcttcttcttctaatcgttctcgtttttagtgatctgccaggttctctctgcacagtgag M T L L L L L I V L V F S D L P G S L C T V S cactgttggagatctgtctgtcctggacggtcagtctgtcactgtcccgtgtcactataacccgcagtacatcagtcacgtgaagtactg T V G D L S V L D G Q S V T V P C H Y N P Q Y I S H V K Y W gtgtcagggccggatgagggagttctgcacgagcctcgcgcgcaccgatgagcccgaatcagcccctaacgggagtggaaaggtgacgat C Q G R M R E F C T S L A R T D E P E S A P N G S G K V T I cgcggacgaccccacgcagcacgtgttcaccgtcagcatgcgcgagctgacggaggaggactcgggctggtacaggtgcggggtggagct A D D P T Q H V F T V S M R E L T E E D S G W Y R C G V E L cggggggatgtgggtggctgacagcactgcctccctgtatatcagcgtcattcaaggtatatcagtggtgagcagtttgcaaagtgcaga G G M W V A D S T A S L Y I S V I Q G I S V V S S L Q S A E ggaaggcagcagcatcactgttcaatgtctctacagtaaaagcctcaggtccagtgagaagcggtggtgtcgcagtggggacttgaactc E G S S I T V Q C L Y S K S L R S S E K R W C R S G D L N S ctgcatggtgacggactctgaaggaaaattcatcagtagcaacgtgttcatgcatgatgacaggaacagcatgttgacggtgacgatgca C M V T D S E G K F I S S N V F M H D D R N S M L T V T M Q gcagctgaagatgagagactcaggctggtactggtgtggagctggtcaacagaaggtggctgttcatgtgtcagtcacaccacaaaccac Q L K M R D S G W Y W C G A G Q Q K V A V H V S V T P Q T T aacactgagcacaacagtcaagaatcaagaatctgtaatgagctcaaatgaccctcacaggcgtcctgtttgggagactcctcttgtggt T L S T T V K N Q E S V M S S N D P H R R P V W E T P L V V gtgtggggtcatattgctggtcctgactgtttttttggcactttggaagttgcggcagttatttaagaataagcagaaacatcgagggac C G V I L L V L T V F L A L W K L R Q L F K N K Q K H R G T aattgaaatgaatgataatctcacaatgtgtccatggagagaaggagactataagaacccctctgtgattttcctgaacgctccagctca I E M N D N L T M C P W R E G D Y K N P S V I F L N A P A Q ggtccagatgctctaaCAGAGGAGCCTCATGATGGGAAACACCAGACTCATGGACATTCATGATGATGAAGAAGATCATAAAGTGATCTG V Q M L * ATGGACACCTTGACTCTTCAAGTCTCTTTATGACGTCTATGAGAGTCCTGCAGACTGGAGAAACTGAAGACTGGTCTAATATTTGACATT CCTTCATATTTAACAGGAAGATCATCTTTCATTGTAATGCATTTTTTTCTTGTCTTGTATTTCCAGTGGTGGACAAAGTACACAAATCAA GTACTTGAGTTAAACTACATATATAATAAAATATTACTCCAGTAAAAAAAAAAAAAAAA

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Fig. 1. Full-length cDNA sequence and amino acid sequence of pIgR in C. auratus. The stop codon is indicated with asterisk (*). The predicted signal peptide is labeled in italics. The Ig-like domain 1 (ILD1) and Ig-like domain 2 (ILD2) are marked in gray shade. The transmembrane domain is marked in bold. The polyadenylation signal (AATAAA) is boxed in the 3ʹ-UTR.

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Fig. 2. Structure profile of C. auratus pIgR as predicted by SMART. Red: Signal peptides; blue: transmembrane domain; IG: Ig-like domain.

3.2. Multiple sequence alignment and phylogenetic analysis

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BLASTp search showed that the deduced amino acid sequence of CapIgR shared higher identity

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with other fish pIgR than with other vertebrate counterparts (Table 2). Amino acid sequence analysis

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and multiple alignment of pIgR revealed that the ILD1 and ILD2 of CapIgR corresponded to

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mammalian pIgR ILD1 and ILD5, respectively. The amino acid sequence of CapIgR contained the

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conserved amino acid residue CWDC, the common key motif KYWC, and DxGxYxC (where x

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stands for any amino acid) in the ILD1 of all vertebrate pIgRs available, and KxWC and

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DxGWYWC in ILD5 (Fig. 3).

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To evaluate the evolutionary relationship of CapIgR with pIgRs from other species, a

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phylogenetic tree was constructed based on the amino acid sequence of pIgRs from 15 vertebrates

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using the neighbor-joining method, the reliability of which was estimated by bootstrapping with

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1000 replications (Fig. 4). As depicted in Fig. 4, fish pIgR, including CapIgR, fell into one cluster,

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whereas the pIgRs in amphibians, avians, and mammals were clustered into another group; moreover,

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CapIgR shared a close relationship with the pIgRs from Sinocyclocheilus anshuiensis and C. carpio.

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Table 2

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Percentage of identity amino acids sequence between CapIgR and pIgRs in the other vertebrates. GenBank Species Identity /% accession No. Anshui Golden-line barbell XP_016298944 83 Common carp ADB97624 78 Zebrafish ABQ10652 71 Rainbow trout ADB81776 51 Sea bass ANZ03107 51 Atlantic salmon ACX44838 50 Turbot AGN54539 50 Olive flounder ADK91435 48 Orange-spotted grouper ACV91878 48 Fugu NP_001266944 47 Atlantic cod AIR74929 41 Human CAA51532 29 Rat EDM09843 28 Mouse AAA67440 28

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ACCEPTED MANUSCRIPT Chicken African clawed frog

AAP69598 ABK62772

28 26

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Fig. 3. Alignment of deduced amino acid sequences of pIgR from C. auratus and other vertebrates. Residues identical in all sequences are shaded in black. Sequences corresponding to signal peptide, ILD1, ILD5,

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transmembrane region, and extracellular region are noted in the brackets at the top of the sequence matrix. The conserved Ig-binding sites in ILD1 of mammals and avians are boxed by dashed lines. CDR-like loops (CDR1, CDR2, and CDR3) in ILD1 of mammals and avians are written in italic and bold type. The conserved amino acid residues in ILD1 are shown in the triangles. The conserved motifs in ILDs are boxed by dashed lines. The ILD2, ILD3, and ILD4 of pIgR are abbreviated in the box in the solid line. The GenBank accession numbers of the aligned sequences are as above in Table 2.

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Fig. 4. Phylogenetic tree constructed with the neighbor-joining method based on the amino acid sequences of pIgR from C. auratus and other vertebrates. The reliability of each node is estimated by bootstrapping with 1000 replications implemented in MEGA 7.0. The scale bar (0.1) represents the genetic distance. The numbers marked near the nodes indicate the bootstrap test scores. The used sequences with the accession no. are listed in Table 2.

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3.3 mRNA expression levels of CapIgR in different tissues

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The mRNA expression levels of pIgR were detected in different tissues of healthy C. auratus by

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qRT-PCR. As shown in Fig. 5, the mRNA expression of pIgR was the most abundant in the liver and

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moderately abundant in the intestine, spleen, and head-kidney. The lowest expression was observed

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in muscle.

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Relative mRNA level

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ACCEPTED MANUSCRIPT Fig. 5. Relative mRNA expression levels of CapIgR in different tissues. The mRNA expression levels of CapIgR in the intestine, skin, gill, liver, spleen, head-kidney, kidney, and muscle were determined by qRT-PCR. For the convenience of comparison, the expression level in muscle was chosen as a calibrator (set as 1). The relative expression of CapIgR was presented as fold change for the calibrator. Data are shown as means ±S.E. (n=3). Different letters above the bars indicate significant difference (P< 0.05).

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3.4 Temporal expression levels of CapIgR after A. hydrophila challenge

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The results of qRT-PCR showed that the temporal expression levels of pIgR in the intestine,

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skin, gill, liver, spleen, and head-kidney of C. auratus infected with A. hydrophila differed among the

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various tissues (Fig. 6).

In the intestine, the mRNA expression level of CapIgR peaked at 6 h post infection (pi) with

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8.29-fold (P < 0.01) relative to the control and then decreased at 12, 24, and 48 hpi to 7.86-, 7.32-,

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and 4-fold, respectively, but remained higher than that in the control (P < 0.01). In the skin, CapIgR

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expression increased at 3 hpi (3.65-fold, P < 0.05) in comparison with the control and then slightly

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increased at 6 hpi (3.54-fold, P < 0.05). Afterward, CapIgR expression suddenly increased, reached

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the maximum level at 12 hpi (5.85-fold, P < 0.01), maintained a high level at 24 hpi (5.15-fold,

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P<0.01), and then sharply reduced to 2-fold at 48 hpi (P < 0.05). In the gill, the mRNA expression

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level of CapIgR gradually increased, peaked at 12 hpi (5.35-fold, P < 0.01), and then drastically

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declined to the normal level at 24 and 48 hpi.

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In the spleen, the mRNA expression of CapIgR exhibited no obvious change at 3 and 6 hpi. It

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drastically increased at 12 hpi (9.04-fold, P < 0.01), peaked at 24 hpi (14.52-fold, P < 0.01), and then

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slightly declined at 48 hpi but still maintained a significantly higher level (9-fold, P < 0.01)

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compared with the control. In the liver and head-kidney, CapIgR expression changes were similar to

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that in the spleen, whereby no obvious change was observed at 3 and 6 hpi compared with the

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control. In the liver, CapIgR expression significantly increased at 12 hpi (5.04-fold), 24 hpi

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(8.55-fold), and 48 hpi (2-fold), with the highest level at 24 hpi (P < 0.01). In the head-kidney, the

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mRNA expression level sharply increased, peaked at 12 hpi (8.59-fold, P < 0.01), and maintained a

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higher level at 24 hpi (8.52-fold, P < 0.01) and 48 hpi (2.12-fold, P < 0.05) compared with the

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control.

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Fig. 6. Temporal expression changes of CapIgR in the intestine, skin, gill, spleen, liver, and head-kidney at 3, 6, 12, 24, and 48 h after A. hydrophila infection. The gene β-actin was used as the internal control to calibrate the cDNA template for all samples. The expression level in the control was set as 1.0, and the results were shown as means ± S.E. (n=3). Compared with the control, significant difference (P < 0.05) was indicated with an asterisk (*), and extremely significant difference (P < 0.01) was signed with two asterisks (**).

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299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317

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4. Discussion

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This study characterized the pIgR gene in C. auratus. The complete cDNA sequence of pIgR

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was 1409 bp. A predicted protein of 327 amino acids in CapIgR was determined to possess the

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typical domain organization of pIgR (i.e., extracellular region with two ILDs, a transmembrane

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region, and an intracellular region), which is in accordance with the structure of fish pIgR reported in

ACCEPTED MANUSCRIPT previous studies [3,8,9,12,16]. BLASTp search showed that the deduced amino acid sequence of

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CapIgR shared a high identity with those of pIgR from other fish, particularly the Cyprinid fish. For

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instance, CapIgR presented 83% sequence identity with Anshui Golden-line barbell, 78% with

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common carp, and 71% with zebrafish. This result indicated a closer relationship between C. auratus

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and the Cyprinid fish as depicted in the phylogenetic tree.

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4.1 Comparison of amino acid sequence and structure of pIgR among vertebrates

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The alignment of multiple amino acid sequences revealed that CapIgR sequence shared a high

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similarity to those from other fish. Although CapIgR presented only 28%–29% sequence identity

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with mammalian pIgR, their primary and secondary structures shared some common features. First,

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similar to mammalian pIgR, CapIgR is also a type І transmembrane glycoprotein, which consists of

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an extracellular region, a transmembrane region, and a cytoplasmic region [2]. Second, CapIgR

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shared several key amino acid residues with other pIgRs, implying a similar function performed by

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the homogenous key amino acid residues. The five key amino acid residues (CWRDC) in the ILD1

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of mammalian pIgR are required for β-pleated sheets to maintain the stability of the Ig fold [26]. In

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the present study, four key amino acid residues (CWDC) in the ILD1 of CapIgR were conserved

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compared with other fish pIgRs, and only one amino acid differed between fish (CWDC) and

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mammalian (CWRDC) pIgR. The CWDC of pIgR could also assist in stabilizing the secondary

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structure in fish. Moreover, pIgR ILD1 harbored the common key motifs KYWC and DxGxYxC (x

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stands for any amino acid) among the vertebrates, and the conserved KxWC and DxGWYWC were

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shown in fish pIgR ILD2 and in mammalian pIgR ILD5.

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Despite the common features of pIgRs between fish and mammal, several crucial functional

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motifs of CapIgR differed from those of mammalian pIgR. First, only two ILDs were present in the

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extracellular region of CapIgR, whereas four were found in chicken and the African clawed frog and

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five (ILD1-5) in mammals [6,7]. Previous studies have shown that the first and second ILDs of fish

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pIgR correspond to the ILD1 and ILD5 of mammalian pIgR [3,8,9,12,16]. In this study, the first and

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second ILDs of CapIgR were highly homologous with mammalian pIgR ILD1 and ILD5,

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respectively. Second, with regard to the difference in glycosylation sites in pIgR between fish and

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other mammals, the human pIgR is a glycosylated protein with seven putative N-glycosylation sites

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in its extracellular region [27,28]; however, in fish, less N-glycosylated sites were predicted. In the

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present study, only one N-glycosylated site was predicted in C. auratus pIgR, which is consistent

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ACCEPTED MANUSCRIPT with the pIgRs in fugu and carp [3,32]; moreover, none are present in the grouper, flounder, and sea

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bass [9,12,16]. Interestingly, ten O-glycosylation residues were predicted in the extracellular region

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of CapIgR. Currently, whether the N-glycosylation of pIgR is essential for its function remains

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controversial. Some studies reported that the glycosylated human SC (secretory component)

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facilitates the transport or release of pIgR into the mucus [29,30]. However, Prinsloo et al. [31]

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reported that N-glycosylation of the recombinant SC is unnecessary for the interaction with

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polymeric immunoglobulins. In fish, less or the absence of N-glycosylated sites in pIgR does not

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affect its binding capability with pIg [9,12,16], implying that N-glycosylation is not required for

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pIgR binding to pIg. Third, in mammals, the highly conserved Ig-binding sites of ILD1 are important

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sites for interaction with dIgA/pIgM, but they have no conserved equivalent regions in CapIgR and

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other fish pIgRs [3,8,9,12]. Moreover, three complementarity-determining region-like loops

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(CDR-like loops) in the ILD1, which plays an important role in forming the binding surface for the

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dIgA/pIgM in mammals [8,33], were not observed in fish. However, the absence of the Ig-binding

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site and the CDR-like loops in ILD1 do not affect the binding of pIgR with pIg in fish. In fish, the

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pIgR combines with IgM in the skin mucus of fugu and flounder [3,12], and with IgM and IgT in the

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gut mucus of trout [10]. In sea bass, the recombinant extracellular domain of pIgR could interact

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with IgG and IgM [16]. Collectively, these results suggest that pIgR demonstrates different

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mechanisms of combining with the pIgs in fish and mammals.

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4.2 CapIgR mRNA expression in various tissues of C. auratus

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The pIgR is widely expressed in various tissues (e.g., intestine, skin, gills, liver, spleen, and

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head-kidney) of many fish (e.g. fugu, carp, grouper, zebrafish, and sea bass) [3,8,9,11-14,16]. In the

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present study, the mRNA expression of pIgR in healthy C. auratus was the most abundant in the liver

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and moderately abundant in the intestine, spleen, and head-kidney. The lowest expression was

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observed in the muscle. The tissue specificity in healthy C. auratus was similar with the previous

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findings in carp, flounder, and sea bass, with the highest expression level in liver [8,12,16]. The liver

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is a vital organ in the immune response, and in the liver of fish, the expression of a large number of

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immune-related genes could be stimulated to improve immune defense; moreover, mucosal IgM in

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fish has been detected in bile ducts and capillaries [8,12,34]. These data suggest that the pIgR in liver

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plays an important role in the transcytosis of mucus IgM from the liver into the bile and subsequently

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into the intestines [8,34]. Notably, the expression levels of pIgR in C. auratus and other fish are not

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ACCEPTED MANUSCRIPT exclusive to mucosal tissues. The expression levels of pIgR in mucosal tissues of the skin and gill are

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often lower compared with those in the liver, spleen and head-kidney; moreover, high pIgR

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transcripts possibly originate from lymphocytes, which are the predominant cells in these lymphoid

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organs [11]. Therefore, apart from mucosal tissues, the liver, spleen, and head-kidney of C. auratus

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should also be investigated for CapIgR mRNA expression in response to A. hydrophila.

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4.3 CapIgR mRNA expression in response to A. hydrophila

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After A. hydrophila challenge, CapIgR expression was significantly upregulated in various

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tissues at most time points, with temporal expression changes fluctuating and presenting in a

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time-dependent manner.

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After the challenge with A. hydrophila, CapIgR expression was upregulated in mucosal tissues

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(intestine, skin and gill) at most time points and presented a changing trend. That is, it initially

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increased but subsequently decreased, which was consistent with the results of pIgR expression

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changes in the hindgut of common carp [17] and in the mucosal tissues of turbot [13] after V.

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anguillarum stimulation. In the present study, the peak value of CapIgR in the different mucosal

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tissues was observed at different time points. In the intestine, the expression of CapIgR sharply

398

reached the peak at 6 hpi; in the skin and gill, the maximum level was reached at 12 hpi. Moreover,

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the highest increase in CapIgR expression during A. hydrophila infection was observed in the

400

intestine. Therefore, we speculated that the immune response of CapIgR is faster and stronger in the

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intestine than in the skin and gill, whereas the immune response of pIgR in turbot is faster and

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stronger in the gill than in the skin and intestine [13]. The different results may be due to the different

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methods of challenge. In the present study, intraperitoneal injection was performed in C. auratus,

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whereas immersion stimulation was performed in turbot. The intraperitoneal injection provided the

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opportunity for the intestines to interact earlier with A. hydrophila; thus, the response was earlier. In

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addition, the significant upregulation of CapIgR mRNA expression was prolonged in the intestine

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and skin (from 3 hpi to 48 hpi). This result can be attributed to the fact that CapIgR could function

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persistently in the intestine and skin of C. auratus to defend against invading bacteria.

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In the current study, CapIgR mRNA expression was induced in the spleen, liver, and

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head-kidney following A. hydrophila infection and exhibited a changing trend similar to that in the

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mucosal tissues. These results were in accordance with the reports in the Atlantic salmon and turbot

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[11,13]. The expression of pIgR is significantly elevated after 14 days in the spleen of Atlantic

ACCEPTED MANUSCRIPT salmon challenged with Lepeophtheirus salmonis [11]. In turbot, the mRNA expression levels of

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pIgR are significantly upregulated in the spleen, liver, and head-kidney after stimulation with V.

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anguillarum [13]. Interestingly, in the present study, the peak value of CapIgR after infection by A.

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hydrophila was higher in the spleen, liver, and head-kidney than in the mucosal tissues. Similarly, the

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peak value of pIgR is higher in the spleen than in the intestine after V. anguillarum stimulation [13].

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Thus, CapIgR plays an important role in immune response to pathogen infection in non-mucosal

419

tissues.

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As previously mentioned, the mRNA expression of CapIgR in various tissues of C. auratus

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initially increased significantly after A. hydrophila stimulation and then decreased. Additionally,

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CapIgR expression in the different tissues generally upregulated at 12 hpi. However, the mRNA

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expression of CapIgR were significantly upregulated in mucosal tissues at the early time points (3 or

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6 hpi). In contrast to those in mucosal tissues, significant increase of CapIgR expression in the spleen,

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liver, and head-kidney only became evident at 12 hpi. In the study, the immune response of CapIgR

426

to A. hydrophila challenge was more rapid in the mucosal tissues than in the liver, spleen, and

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head-kidney. Therefore, CapIgR in the mucosal tissues may play a pivotal role in the early stages of

428

A. hydrophila challenge. The drastic and persistent upregulation of CapIgR in the various tissues

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contributed to the transport of pIgs in order to protect organisms from A. hydrophila invasion.

430

Although the significant inductions of CapIgR in the spleen, liver, and head-kidney emerged at the

431

middle time point of A. hydrophila infection, the peak value of CapIgR was higher in the

432

non-mucosal tissues (spleen, liver, and head-kidney) than in the mucosal tissues. Therefore, CapIgR

433

in the non-mucosal tissues may play a more important role in the middle or late stage of A.

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hydrophila infection. Collectively, CapIgR plays various roles in bacterial infection in different

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tissues and at different time points.

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In conclusion, the full-length cDNA sequence of CapIgR was cloned and characterized in C.

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auratus. The deduced protein CapIgR presented a typical structure of pIgR and contained the

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conserved amino acid residues and common key motifs in pIgRs, which was suggested to perform a

439

similar function to corresponding pIgRs in other fish. In healthy C. auratus, the mRNA expression

440

levels of CapIgR were ubiquitous in the eight tissues examined, with the highest expression in the

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liver. The temporal expression pattern of CapIgR following A. hydrophila challenge was first

442

investigated. Although the immune response of CapIgR was different in the various tissues and at

ACCEPTED MANUSCRIPT different time points, CapIgR expression levels were generally upregulated at 12 hpi. Furthermore,

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CapIgR in the mucosal tissues (intestine, skin, and gill) might play a pivotal role in the early stage of

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A. hydrophila challenge, whereas CapIgR response in the non-mucosal tissues (liver, spleen, and

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head-kidney) occurred in the middle or late stage of A. hydrophila challenge. In brief, CapIgR

447

presents a rapid immune response to A. hydrophila challenge and plays an important role in the

448

immune defense of fish. The findings of this study contribute to understanding the important role of

449

CapIgR in the immune response of Qihe crucian carp and offers new perspectives in protecting fish

450

from pathogen infection.

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Acknowledgments

This study was sponsored by the Joint Fund of Natural Science Foundation of China and Henan

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Province (Project No. U1604104) and Program for Innovative Research Team in Science and

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Technology in the University of Henan Province (Project No. 15IRTSTHN018). The authors would

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like to thank their colleagues for the valuable suggestions on the overall manuscript preparation.

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Highlights • The full-length CapIgR cDNA was cloned and characterized in Carassius auratus. • CapIgR was ubiquitously expressed in various tissues with the highest in liver.

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• CapIgR transcriptions were mostly up-regulated after A. hydrophila challenge.

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• CapIgR expressions fluctuated in a time-dependent manner after bacterial stress.