The role of oncoprotein NM23 gene from Exopalaemon carinicauda is response to pathogens challenge and ammonia-N stress

Accepted Manuscript The role of oncoprotein NM23 gene from Exopalaemon carinicauda is response to pathogens challenge and ammonia-N stress Yafei Duan, Jitao Li, Jian Li, Qianqian Ge, Zhe Zhang, Ping Liu PII:

S1050-4648(15)30108-X

DOI:

10.1016/j.fsi.2015.08.018

Reference:

YFSIM 3571

To appear in:

Fish and Shellfish Immunology

Received Date: 30 June 2015 Revised Date:

17 August 2015

Accepted Date: 20 August 2015

Please cite this article as: Duan Y, Li J, Li J, Ge Q, Zhang Z, Liu P, The role of oncoprotein NM23 gene from Exopalaemon carinicauda is response to pathogens challenge and ammonia-N stress, Fish and Shellfish Immunology (2015), doi: 10.1016/j.fsi.2015.08.018. 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.

ACCEPTED MANUSCRIPT 1

The role of oncoprotein NM23 gene from Exopalaemon carinicauda is response to

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pathogens challenge and ammonia-N stress

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Yafei Duana, Jitao Lib, Jian Lib, Qianqian Geb, Zhe Zhanga, Ping Liub*

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a

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Agriculture, South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences,

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Guangzhou 510300, PR China

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b

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Fisheries Research Institute, Chinese Academy of Fishery Sciences, 106 Nanjing Road, Qingdao

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266071, PR China

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Key Laboratory of South China Sea Fishery Resources Exploitation & Utilization, Ministry of

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Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture, Yellow Sea

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ABSTRACT Oncoprotein NM23, as a family of genes encoding the nucleoside diphosphate (NDP)

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kinase, plays important roles in bioenergetics, DNA replication, differentiation and tumor metastasis. In

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this study, a full-length cDNA of NM23 (designated EcNM23) was cloned from Exopalaemon

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carinicauda by using rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of

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EcNM23 was 755 bp, which contains an open reading frame (ORF) of 518 bp, encoding a 175

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amino-acid polypeptide with the predicted molecular weight of 19.60 kDa and estimated isoelectric

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point of 7.67. The deduced amino acid sequence of EcNM23 shared high identity (86%-93%) with that

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of other crustaceans. a NDP kinase super family signature was identified in E. carinicauda EcNM23.

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Quantitative real-time RT-qPCR analysis indicated that EcNM23 was expressed in all the examined

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tissues with the high expression level in hemocytes and ovary. The EcNM23 expression in

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immune-related tissues changed rapidly and reached peak at different time after pathogens (Vibrio

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* Corresponding author. Tel.: +86 532 85836605; fax: +86 532 85826690. E-mail address: [email protected] (P. Liu).

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parahaemolyticus and WSSV) challenge and ammonia-N stress treatment. The results suggested that

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EcNM23 might be associated with the immune defenses to pathogens infection and ammonia-N stress

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in E. carinicauda.

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Keywords: Exopalaemon carinicauda, Oncoprotein NM23, Vibrio parahaemolyticus, White spot syndrome virus (WSSV), Ammonia-N stress

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The ridgetail white prawn Exopalaemon carinicauda is an economically important shrimp species

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naturally distributed in the coasts of the Yellow Sea and the Bohai Sea, China, which contributes to one

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third of the gross output of the polyculture ponds in eastern China [1]. Due to its commercial value,

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“milky shrimp” disease caused by Hematodinium [2], immune gene discovery by expressed sequence

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tags (ESTs) [3] and transcriptome analysis [4], and identification of immune-related genes such as heat

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shock protein (HSP90) [5], selenium dependent glutathione peroxidase (GPx) [6] and calreticulin (CRT)

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[7] have been studied in E. carinicauda. However, with the development of intensive culture and the

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ecologic environmental deterioration, various diseases caused by pathogens and environmental stresses

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have blossomed within booming in cultured shrimp populations, causing economic losses to

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commercial shrimp aquaculture [6,7]. Previous studies have demonstrated that the suboptimal

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environmental conditions could affect the immunity of E. carinicauda, for example, pH and ammonia

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stress could cause affect the immune response of HSP90 [5], pathogens challenge could induce the

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immune-related genes such as GPx [6] and CRT [7]. Therefore, better understanding of the innate

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immune abilities and immune defense mechanisms of shrimp will be beneficial to the development of

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health management in shrimp aquaculture.

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ACCEPTED MANUSCRIPT Biosynthesis of non-adenine nucleoside triphosphates is critical for bioenergetics, DNA replication,

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sugar and lipid biosynthesis, and signal transduction pathways, and this function is achieved by

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nucleoside diphosphate (NDP) kinase (EC 2.7.4.6) [8,9]. Oncoprotein NM23 is a functional NDP

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kinase, which catalyzes phosphoryl transfer c-phosphate from nucleoside triphosphate (NTP) to

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diphosphate (NDP) [10,11], and involved in several cellular functions, such as the control of

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transcription, DNA repair and cellular proliferation, differentiation, apoptosis, and invasion suppression

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[12,13]. NM23 was initially identified as a potential tumor metastasis inhibitor, with subsequent studies

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showing that it belongs to a structurally large and functionally conserved gene family [14]. The NM23

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family of proteins can be separated into two groups based on their sequence homology, such as group I

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(NM23-H1 to H4) and group II (NM23-H5 to H8) [15]. The NM23 genes of group I possesses the

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classic enzymatic activity of NDP kinase, however, only one product of the group II genes, NM23-H6,

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has been demonstrated to catalyze the NDP kinase reaction [16]. In crustaceans, only one isoform of

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NDP kinase has been reported [11,12,17], and has documented functions in various stress and immune

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response, for example, NM23 transcripts were increased significantly in Eriocheir sinensis after Vibrio

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anguillarum challenge [12], and up-regulated upon WSSV challenge in Litopenaeus vannamei [8,17].

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Previous studies have demonstrated that the suboptimal environmental conditions, such as ammonia

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nitrogen (ammonia-N) stress could affect the immunity [18,19], growth and molting [20], oxygen

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consumption and ammonia excretion of crustaceans [21]. Vibrio parahaemolyticus and WSSV caused

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the most serious disease leading to major losses in the shrimp aquaculture industry around the world

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[7,22]. For example, acute hepatopancreatic necrosis disease (AHPND), also known as early mortality

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syndrome (EMS), which causative agents was V. parahaemolyticus, has caused large scale losses in

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farmed shrimp production [22-24], influenced the antioxidative status and caused oxidative stress and

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ACCEPTED MANUSCRIPT tissue damage via confusion of antioxidant enzymes [25], and induced the expression level of c-Fos

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and c-Jun gene in shrimps [26]. So far there are still no effective preventive and therapeutic measures

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against AHPND/EMS diseases. Shrimps lack an adaptive immune system and their defense

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mechanisms mainly rely on innate immune responses for protecting them against invaders and the

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environmental stresses. Nucleoside kinases are antiviral targets of several pathogens, because they can

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process the activation of nucleotide or nucleoside analogues [8]. However, none is known about the

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potential role of EcNM23 in the ridgetail white prawn E. carinicauda against pathogens challenge

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(such as V. parahaemolyticus and WSSV) and ammonia-N stress.

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The aim of this study was to clone the full-length cDNA of NM23 from hemocytes of E. carinicauda,

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compare its sequence with other known NM23s, investigate the expression pattern of EcNM23 in

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various tissues of E. carinicauda, and evaluate its expression in immune-related tissues of E.

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carinicauda after pathogens (V. parahaemolyticus and WSSV) challenge and ammonia-N stress. These

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results will be essential to understand the role of EcNM23 in immune response against pathogens

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challenge and ammonia-N stress in E. carinicauda.

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2. Materials and methods

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2.1. Animals materials

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Healthy adult E. carinicauda, averaging weight 1.33 ± 0.32 g, were collected from a commercial

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farm in Qingdao, China. They were cultured in 200 L polyvinyl chloride polymer (PVC) tanks with

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filtered aerated seawater (salinity 30‰, pH 8.2) at 22 ± 0.5 ℃ for 7 days before processing. There were

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100 shrimps in each group. The shrimps were fed daily with a ration of 10% of body weight, and

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two-thirds of the water in each group was renewed once daily.

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2.2. RNA extraction and cDNA synthesis

ACCEPTED MANUSCRIPT Hemocytes were collected with a syringe which contained an equal volume of anti-coagulant buffer

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(1.59 g sodium citrate, 3.92 g sodium chloride, 4.56 g glucose, 0.66 g EDTA-2Na, 200 mL ddH2O) [27],

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and centrifuged at 800 g, 4 ℃ for 15 min. Total RNA was extracted from hemocytes using Trizol

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Reagent (Invitrogen, USA) following the manufacturer’s protocol. The RNA samples were analyzed in

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1.0% agarose electrophoresis and quantitated at 260 nm, all OD260/OD280 were between 1.8 and 2.0.

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The 3’ and 5’ ends RACE cDNA template were synthesized using SMARTTM cDNA Kit (Clontech,

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USA) following the protocol of the manufacturer.

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2.3. Cloning the full-length cDNA of EcNM23

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An EST sequence corresponding to NM23 was obtained from E. carinicauda hemocytes cDNA

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library of our laboratory (GenBank accession no. JK996340), and has been reported by Duan et al.

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(2013) [3]. BLAST analysis showed that it has high identities with NM23s of other crustaceans.

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According to the EST sequence, a gene specific primer F1 was designed for 3’ RACE, and primer R1

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was designed for 5’ RACE (Table 1) and its 3’ and 5’ ends were obtained using SMART RACE cDNA

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Amplification Kit (Clontech, USA). For 3’ RACE, the PCR reaction was performed using the primer

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F1 and the anchor primer UPM (Table 1). The 50 µL PCR reactions contained the cDNA 2.5 µL of

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template, 10 × Advantage 2 PCR buffer 5 µL, dNTP Mix (10 µmol/L) 1 µL, 50 × Advantage 2

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Polymerase Mix 1 µL, primer UPM (10 µmol/L) 5 µL, primer F1 (10 µmol/L) 1 µL, PCR-Grade water

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34.5 µL. The PCR reaction conditions were 5 cycles of 94 ℃ for 30 s, 72 ℃ for 3 min, 5 cycles of

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94 ℃ for 30 s, 70 ℃ for 30 s, and 72 ℃ for 3 min, and 25 cycles of 94 ℃ for 30 s, 68 ℃ for 30 s and

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72 ℃ for 3 min. For 5’ RACE, the PCR reaction was performed using the primer R1 and the anchor

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primer UPM (Table 1). The PCR reaction systems and conditions were the same as described above.

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The PCR fragments were subjected to electrophoresis on 1.5% agarose gel to determine length

ACCEPTED MANUSCRIPT differences, and the target band was purified by PCR purification kit (Promega, USA). The purified

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products were cloned into PMD18-T vector, following the instructions provided by the manufacturer

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(TaKaRa, Japan). Recombinant bacteria were identified by blue/white screening and confirmed by PCR.

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Plasmids containing the insert were purified (Promega minipreps) and used as a template for DNA

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sequencing.

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2.4. Sequence analysis

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The nucleotide and deduced amino acid sequences of EcNM23 cDNA were analyzed and compared

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using the BLAST search programs (http://www.blast.ncbi.nlm.nih.gov/Blast.cgi). The signal peptide

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was predicted by SignalP program (http://www.cbs.dtu.dk/services/SignalP/). The multiple sequence

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alignment of NM23 amino acid sequences was performed using the programs of Vector NTI advance

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10.3 (Invitrogen). A phylogenetic NJ tree of NM23s was constructed by the MEGA 4.0 software [28].

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2.5. Tissue expression of EcNM23

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Hemocytes, hepatopancreas, gill, muscle, ovary, intestine, stomach and heart were dissected from

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unchallenged E. carinicauda. The mRNA expression levels of EcNM23 in different tissues were

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determined by quantitative real-time RT-qPCR. Total RNA was extracted as described above. The RNA

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samples were analyzed in 1.0% agarose electrophoresis and quantitated at 260 nm, all OD260/OD280

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were between 1.8 and 2.0. Total RNA (5 µg) was reverse transcribed using the PrimeScriptTM Real time

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PCR Kit (TaKaRa, Japan) for real-time quantitative RT-qPCR analysis. The 18S rRNA of E.

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carinicauda (GenBank accession number: GQ369794) was used as an internal control for expression.

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2.6. Experimental design of V. parahaemolyticus and WSSV challenge

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The experiments were divided into the bacterial (V. parahaemolyticus) challenged group, the virus

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(WSSV) challenged group and the control group, and there were three replicates in each group,

ACCEPTED MANUSCRIPT respectively. V. parahaemolyticus strains and WSSV crude extract were provided by the Mariculture

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Disease Control and Pathogenic Molecular Biology Laboratory, Yellow Sea Fisheries Research

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Institute. V. parahaemolyticus strains (no. 20130628001S02) was obtained from the AHPND-infected

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L. vannamei and activated on marine agar 2611E. WSSV crude extract was obtained from 10 g

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WSSV-infected gills from L. vannamei, the gills were homogenized separately in 10 mL sterile 0.9%

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saline solution and centrifuged at 1200 rpm, 4 ℃ for 20 min, and then the supernatant was filtered

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through a 0.45 µm filter. Quantitative detection of WSSV was performed by RT-qPCR on an ABI

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PRISM 7500 Sequence Detection System (Applied Biosystems, USA), the methods referred to Durand

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and Lightner [29]. The RT-qPCR was carried out in a total volume of 20 µL, containing 12.5 µL of

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Perfect Real Time premix (1×) (RR039A, TaKaRa), 50-100 ng of virus DNA, 0.25 µmol/L each of F3

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and R3 primer, 0.125 µmol/L of probes, and added sterile ddH2O to total volume of 20 µL.

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Recombinant plasmid PUCm-T/WSSV69 containing purpose fragment was used as standard. The PCR

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program was 94 ℃ for 10 s, then 40 cycles of 95 ℃ for 5 s and 60 ℃ for 34 s.

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For V. parahaemolyticus challenged group, shrimps were injected individually with 20 µL live V.

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parahaemolyticus suspension (1 × 109 CFU/mL) in sterile 0.9% saline solution, resulting in 2 × 107

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CFU shrimp-1. For WSSV challenged group, shrimps were injected individually with 20 µL live WSSV

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crude extract (106 copies/mL) as described above. The control group received individually an injection

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of 20 µL sterile 0.9% saline solution. Then both the challenged and control shrimps were returned to

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the PVC tanks of aerated seawater and fed at 25 ℃ as described above. The seawater waste was dealt

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with safely to make it harmless for environment. Through the separation and identification of

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pathogens from shrimps in the V. parahaemolyticus and WSSV challenged group, respectively, the

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results showed that the shrimp was infected with particular pathogen. Hemocytes and hepatopancreas

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ACCEPTED MANUSCRIPT of three shrimps from each treatment (the challenged group and the control group) were randomly

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sampled at 0, 3, 6, 12, 24, 48 and 72 h post-injection respectively, then the samples were snap-frozen in

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liquid nitrogen. There were three replicates for each time point. Total RNA was extracted and the first

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strand cDNA was synthesized as described above.

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2.7. Experimental design of ammonia-N stress

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The median lethal of total ammonia-N were found to be 140.28 mg/L for 72 h to adult E.carinicauda

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[30]. Based on these datas, four different ammonia-N concentrations treatment were designed in the

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preliminary experiment to confirm the suitable concentration of ammonia-N stress, such as 17.54,

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35.07, 70.14 and 140.28 mg/L. Ultimately, the preliminary experiment results indicated that the

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survival rate of shrimps was 70% after 72 h and was enough to take samples when the ammonia-N

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concentration was 35.07 mg/L. Therefore, the ammonia-N concentration of 35.07 mg/L was chosen for

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ammonia-N stress in the present study.

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In the ammonia-N stress tests, two different ammonia-N concentrations treatment of 0 (normal

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seawater as control) and 35.07 mg/L were adjusted with diluting 10 g/L NH4Cl. During the experiment,

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the ammonia-N concentration of the seawater were measured every 6 h by hypo-bromate oxidimetry

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method [31]. The water was renewed daily and NH4Cl was added to keep the concentration of

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ammonia-N as 35.07 mg/L. For each treatment, hepatopancreas and gills from three individuals were

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sampled at 0, 3, 6, 12, 24, 48 and 72 h after ammonia-N exposure, then the samples were snap-frozen

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in liquid nitrogen. There were three replicates for each time point. Total RNA was extracted and the

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first strand cDNA was synthesized as described above. After the ammonia-N stress experiment, the

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seawater waste was dealt safely with nitrifying bacteria and denitrifying bacteria to make it harmless

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for environment.

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2.8. Expression of EcNM23 after pathogens challenge and ammonia-N stress Real time quantitative RT-qPCR was performed on an ABI PRISM 7500 Sequence Detection System

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(Applied Biosystems, USA) to investigate the expression of EcNM23. The pair of specific primers F2

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(10 µmol/L) and R2 (10 µmol/L) (Table 1) was used to amplify a PCR product of 100 bp. Two primers

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18S-HF (10 µmol/L) and 18S-HR (10 µmol/L) (Table 1) were used to amplify an 18S rRNA gene of

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147 bp as an internal control to verify the successful reverse transcription and to calibrate the cDNA

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template. The RT-qPCR was carried out in a total volume of 20 µL, containing 10 µL SYBR® Premix

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Ex TaqTM Ⅱ (2×) (TaKaRa), 2 µL of the 1:5 diluted cDNA, 0.8 µL each of F2 and R2 primer (or

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18S-HF and 18S-HR to amplify the 18S), 0.4 µL ROX Reference Dye Ⅱ (50×)*3 and 6 µL

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DEPC-treated water. The PCR program was 95 ℃ for 30 s, then 40 cycles of 95 ℃ for 5 s and 60 ℃

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for 34 s, followed by 1 cycle of 95 ℃ for 15 s, 60 ℃ for 1 min and 95 ℃ for 15 s. DEPC-treated water

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for the replacement of template was used as negative control.

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RT-qPCR data from three replicate samples were analyzed with the ABI 7300 system SDS Software

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(Applied Biosystems, USA), for estimating transcript copy numbers for each sample. The comparative

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CT method was to analyze the relative expression levels of EcNM23. The CT for the target amplified

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products of EcNM23 and internal control 18S rRNA was determined for each sample. The difference in

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the CT between the target and the internal control, called △CT, was calculated to normalize the

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differences in the amount of template and the efficiency of the RT-qPCR. In the same challenge time,

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the △CT of the control group was used as the calibrator, and the difference between the △CT of the

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challenged group and the control group was called △△CT. The expression level of EcNM23 was

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calculated by the 2-△△CT comparative CT method [32]. Statistical analysis was performed using SPSS

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software (Ver 11.0). Statistical significance was determined using one-way ANOVA [33] and post hoc

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Duncan multiple range tests. Significance was set at P < 0.05.

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3. Results

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3.1. Molecular characterization of EcNM23 The full-length EcNM23 cDNA of E. carinicauda was obtained by 5’ and 3’ RACEs, and the results

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were shown in Fig. 1. The full-length of EcNM23 was 755 bp, containing a 528 bp open reading frame

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(ORF). The cDNA contained a 5’-untranslated region (UTR) of 24 bp, a 3’-UTR of 203 bp including a

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stop codon (TAA), polyadenylation signal (AATAA) and a poly A tail. The EcNM23 cDNA sequence

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and deduced amino acid sequence has been submitted to the GenBank (GenBank accession number:

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KP684055).

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The ORF of EcNM23 encoded 175 amino acids without signal peptide analyzed by SignalP software.

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The calculated molecular mass was 19.60 kDa, and the estimated isoelectric point was 7.67. SMART

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program analysis revealed that EcNM23 had one conserved NDP kinase super family signature

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(NIIRGSD) (Asn138-Asp144) and it was a typical NDP kinase active site motif.

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3.2. Multiple sequences and phylogenetic analysis

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Sequence analysis with the BLASTP program revealed that the deduced amino acid sequence of

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EcNM23 exhibited identities with NM23 of invertebrates and vertebrates. It displayed high identities

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with NM23s of Macrobrachium rosenbergii (93%), P. monodon (91%), L. vannamei (90%), E. sinensis

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(89%), Scylla paramamosain (86%), Ictalurus punctatus (76%), Danio rerio (76%), Columba livia

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(76%), Astyanax mexicanus (75%), Chelonia mydas (75%), Bos taurus (75%), Homo sapiens (75%)

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and Gallus gallus (74%) counterparts, respectively. Multiple sequence alignment of EcNM23 with

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other known NM23s revealed that they were highly conserved, especially in the regions of one NDPK

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super family signatures (Fig. 2).

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ACCEPTED MANUSCRIPT Based on the sequences of NM23s, a Neighbor-Joining phylogenetic tree was constructed using

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MEGA 4.0 (Fig. 3). Two main groups, group I (H1, H2, H3, H4) and group II (H5, H6, H7, H8, H9,

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H10) were separated in the tree. The known NM23s from crustaceans were classified into two

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subgroups: NM23 of E. carinicauda, M. rosenbergii, P. monodon, L. vannamei and E. sinensis were

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clustered into H1 and H2 subgroups, and showed closer evolutionary relationships with H1. EcNM23

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of E. carinicauda showed higher homology with NM23 of M. rosenbergii.

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3.3. Tissue expression of EcNM23

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Quantitative real-time RT-qPCR was employed to investigate the expression profiles of EcNM23

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mRNA in different tissues, such as hemocytes, hepatopancreas, gill, muscle, ovary, intestine, stomach

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and heart (Fig. 4). The mRNA transcripts of EcNM23 could be detected in all the examined tissues

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with different expression levels. The highest expression level of EcNM23 was found in hemocytes and

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ovary, and the lowest was in gill and stomach.

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3.4. Cumulative survival of E. carinicauda after pathogens challenge and ammonia-N stress

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The cumulative survival of E. carinicauda after pathogens challenge and ammonia-N stress were

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shown in Fig. 5. In the V. parahaemolyticus challenged group, the death symptoms was observed at 6 h

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and shrimps abundantly died at 12 h (mortality rate was 12%), then died gradually at 24-72 h (mortality

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rate was 59% at 72 h). In the WSSV challenged group, there were no obvious infection symptoms at

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0-6 h, and shrimps gradually died at 12-72 h (mortality rate was 70% at 72 h). In the ammonia-N stress

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group, the infection symptoms was observed at 3 h, then the cumulative mortality rates increased

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gradually in the first 48 h after stress (mortality rate was 33.33% at 48 h). The serious infection

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symptoms included weakness vitality, slowness reaction, lessen ingestion, abundantly died, and so on.

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However, there were no shrimp died in the control group.

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3.5. mRNA expression of EcNM23 in hemocytes and hepatopancreas after V. parahaemolyticus

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challenge The mRNA expression level of EcNM23 in hemocytes and hepatopancreas of E. carinicauda after V.

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parahaemolyticus challenge were shown in Fig. 6. Compared to the control group, the expression of

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EcNM23 in hemocytes increased significantly and reached the highest level at 12 h (4.84-fold of the

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control group, P < 0.05), then it decreased gradually and reached to the control group level at 48 h (Fig.

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6a). EcNM23 transcripts in hepatopancreas obviously decreased to the lowest level at 3 h (0.66-fold of

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the control group, P < 0.05), then it increased to a peak value at 6 h (1.42-fold of the control group, P <

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0.05) which was higher than the control, and maintained a high level at 24 and 72 h (Fig. 6b).

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3.6. mRNA expression of EcNM23 in hemocytes and hepatopancreas after WSSV challenge

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Expression profiles of EcNM23 mRNA in hemocytes and hepatopancreas of E. carinicauda after

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WSSV challenge were shown in Fig. 7. Compared with the control group, EcNM23 transcripts in

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hemocytes were up-regulated significantly and reached to the peak level at 12 h (3.48-fold of the

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control group, P < 0.05), then it down-regulated gradually from 24 h to 72 h, but it was still

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significantly higher than the control (P < 0.05) (Fig. 7a). In hepatopancreas, the EcNM23 mRNA

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expression levels increased significantly to the maximum at 6 h (4.59 -fold of the control group, P <

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0.05), after a decrease from 12 h to 24 h, it decreased to the minimum at 48 h (0.57-fold of the control

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group, P < 0.05) (Fig. 7b).

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3.7. mRNA expression of EcNM23 in hepatopancreas and gills after ammonia-N stress

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The expression pattern of EcNM23 mRNA in hepatopancreas and gills of E. carinicauda after

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ammonia-N stress were shown in Fig. 8. Compared with the control group, EcNM23 transcripts in

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hepatopancreas were no significant changes from 3 h to 12 h (P ＞ 0.05), then it induced significantly

ACCEPTED MANUSCRIPT at 24 h and reached the highest at 72 h (3.61-fold of the control group, P < 0.05) (Fig. 8a). However,

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the expression of EcNM23 in gills showed a trend different from that in hepatopancreas. The level of

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EcNM23 transcripts in gills was reduced significantly from 3 h to 12 h, then it increased gradually to

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the peak level at 72 h (2.19-fold of the control group, P < 0.05) (Fig. 8b)

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4. Discussion

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The identification and characterization of immune-related genes are essential for the elucidation of

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immune defense mechanisms and disease control. In the present study, a new full-length 1725 bp of

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NM23 gene which named EcNM23, was isolated and characterized from the ridgetail white prawn E.

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carinicauda. The deduced amino acid sequence of EcNM23 showed high identities (93%-86%) with

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NM23s of other crustaceans. Multiple sequence alignment analysis revealed that the typical NDP

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kinase signature motifs of E. carinicauda EcNM23 were highly conserved (Fig. 2). Phylogenetic

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analysis further suggested that EcNM23 of E. carinicauda was clustered with NM23s of other

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crustaceans and molluscs into the NM23-H1 subgroup (Fig. 3).

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Quantitative real-time RT-qPCR revealed that EcNM23 was widely distributed in all the examined

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tissues of E. carinicauda, and abundant in hemocytes (Fig. 4). Hemocytes were regarded as the most

279

important immune organ in invertebrates and play a crucial role in the host immune functions when the

280

organism is attacked by microorganisms or viruses [6]. EcNM23 was constitutively expressed in

281

various tissues of E. carinicauda suggesting its multifunction in several biological processes, and one

282

of its main functions involves immune response mechanisms. In addition, the high expression level of

283

EcNM23 in ovary indicated that it might be related to gonad development of shrimps. Previous studies

284

have demonstrated that NM23 has potential roles in spermiogenesis and early differentiation of oocyte

285

in M. rosenbergii through the situ hybridization of gonad sections [11].

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ACCEPTED MANUSCRIPT Virulent pathogens are an important impact factor to aquaculture. V. parahaemolyticus and WSSV

287

are both the extremely virulent pathogen prevalent causing mass mortalities and economic losses in

288

shrimp aquaculture [34-36]. When pathogens enter into the body of the shrimp, they will encounter the

289

innate immune systems [37]. Hemocytes and hepatopancreas are the main cells and tissue involved in

290

the immune response, and the major site for the synthesis of immune defense molecules involved in

291

eliminating pathogens or other particulate matter in crustaceans [38-40]. Information on the expression

292

profile of the EcNM23 gene in hemocytes and hepatopancreas after V. parahaemolyticus and WSSV

293

challenge would be helpful to better understand its immunological function. In the present study, in

294

order to know whether EcNM23 is related to the immune response of shrimp, live V. parahaemolyticus

295

and WSSV were chosen to challenge the shrimp, so that the shrimp health condition could be affected

296

severely by the production of pathogens.

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The effect of V. parahaemolyticus to the expression of NM23 has not been evaluated in crustaceans.

298

In our study, the expression of EcNM23 in hemocytes and hepatopancreas increased significantly at 6 h

299

and 24 h respectively, indicating that EcNM23 could be induced by V. parahaemolyticus. As time

300

progressed, the expression of EcNM23 decreased significantly at 24 h in hemocytes, which might

301

because of the V. parahaemolyticus infection progress brought by more bacterias, and destroyed

302

severely to the normal function of shrimp's cells and finally caused that the expression of EcNM23 in

303

the challenged group decreased gradually [41]. The EcNM23 expression profiles were different in

304

hemocytes and hepatopancreas in response to V. parahaemolyticus challenge, which might be due to

305

the different cells and tissues function in the immune defense system [42]. Our findings were consistent

306

with those previously reported in other crustaceans. After E. sinensis infection by V. anguillarum, peak

307

levels expression of NM23 were induced at 8 h and 6 h in hepatopancreas and gills respectively, then

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ACCEPTED MANUSCRIPT 308

levels of expression declined gradully after 12 h infection [12]. The expression profiles of NM23 with

309

vibriosis challenge indicated that it was immunoresponsive and might be involved in the immune

310

response against vibriosis infection in shrimps. WSSV is a large DNA virus with a broad host range among shrimps and the causative agent of

312

White Spot disease of shrimp, causing mass mortalities and economic losses in shrimp aquaculture

313

[43,44]. Previous studies have demonstrated that the biosynthesis of DNA replication is achieved by

314

NDP kinase, and the maintenance of the deoxynucleotide triphosphate (dNTP’s) pool is key for DNA

315

replication to occur [45,46]. During viral DNA replication, a dNTP’s pool is required and alteration of

316

the nucleotide supply is an antiviral strategy used against viral diseases [8,47]. Quintero-Reyes et al.

317

(2013) have demonstrated shrimp NM23 is a functional NDP kinase, and its activity and affinity

318

towards deoxynucleoside diphosphates [8]. Therefore, NM23 might have potential function in blocking

319

WSSV viral replication. In addition, the NM23 mRNA expression levels were up-regulated when L.

320

vannamei upon viral infection [17]. Similar result was also found in another transcriptome study from

321

WSSV-challenged organisms [48]. In this study, the expression levels of NM23 increased in hemocytes

322

and hepatopancreas at 3 h and 6 h post-WSSV infection. These results all implied that NM23 might be

323

involved in anti-virus immune system of shrimp.

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324

Ammonia-N is the principal end product of protein catabolism, which is one of the important

325

environmental toxic factors in pond [49]. Elevated concentrations of ammonia can cause impairment in

326

numerous organs and induce a modification of the immune system in aquatic animals [5,50]. Gills are

327

the respiratory organs of crustaceans and contacted directly with the seawater, so the environmental

328

toxic factors changes might affect in the physiological function or induce injury to this organ more so

329

than other organs. Therefore, information on the transcript level of the EcNM23 gene in hepatopancreas

ACCEPTED MANUSCRIPT and gills after ammonia-N stress would be essential to better understand its regulatory mechanism. In

331

the present study, the transcript level of EcNM23 in gills decreased at 3-12 h post ammonia-N stress,

332

which suggested that high concentration of ammonia-N exposure (35.07 mg/L) caused a depression in

333

the immune responses of E. carinicauda. Similar studies also have been reported that the expression of

334

the prophenoloxidase and peroxinectin gene in Penaeus stylirostrisir decreased respectively by 60%

335

and 50% in response to the ammonia-N stress [51]. After E. carinicauda exposed to ammonia-N stress

336

48 h, the expression levels of EcNM23 increased till the end of the experiment, which could be

337

speculated that the increase of EcNM23 mRNA under ammonia-N stress presumably reflected the

338

cellular requirement for more of EcNM23 protein to repair damaged proteins, and the immune-related

339

genes such as NM23 and Relish attempted to participate in the progress of resisting the ammonia-N

340

stress, but fail to recover the physiological status of shrimps [52].

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Acknowledgments

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The authors are grateful to all the laboratory members for experimental material preparation and

344

technical assistance. This study was supported by the earmarked fund for National ‘‘863’’ Project of

345

China (No. 2012AA10A409), Modern Agro-industry Technology Research System (No. CARS-47),

346

National Natural Science Foundation of China (No. 31472275), Special Fund for Agro-scientific

347

Research in the Public Interest (No. 201103034), Guangdong Provincial Special Fund for Marine

348

Fisheries Technology (A201501B15), and Special Scientific Research Funds for Central Non-profit

349

Institutes, South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences

350

(2014TS15).

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ACCEPTED MANUSCRIPT References

353

[1] J.T. Li, P. Ma, P. Liu, P. Chen, J. Li, The roles of Na+/K+-ATPase α-subunit gene from the ridgetail

354

white prawn Exopalaemon carinicauda in response to salinity stresses, Fish Shellfish Immunol. 42

355

(2014) 264-271.

356 357

RI PT

352

[2] W.J. Xu, J.J. Xie, H. Shi, C.W. Li, Hematodinium infections in cultured ridgetail white prawns, Exopalaemon carinicauda, in eastern China, Aquaculture 300 (2010) 25-31.

[3] Y.F. Duan, P. Liu, J.T. Li, J. Li, P. Chen, Immune gene discovery by expressed sequence tag (EST)

359

analysis of hemocytes in the ridgetail white prawn Exopalaemon carinicauda, Fish Shellfish

360

Immunol. 34 (2013) 173-182.

M AN U

SC

358

[4] J.T. Li, J. Li, P. Chen, P. Liu, Y.Y. He, Transcriptome analysis of eyestalk and hemocytes in the

362

ridgetail white prawn Exopalaemon carinicauda: assembly, annotation and marker discovery, Mol.

363

Biol. Rep. 42 (2015) 135-147.

TE D

361

[5] J.T. Li, J.Y. Han, P. Chen, Z.Q. Chang, Y.Y. He, P. Liu, et al., Cloning of a heat shock protein 90

365

(HSP90) gene and expression analysis in the ridgetail white prawn Exopalaemon carinicauda, Fish

366

Shellfish Immunol. 32 (2012) 1191-1197.

368 369

[6] Y.F. Duan, P. Liu, J.T. Li, J. Li, P. Chen, Expression profiles of selenium dependent glutathione

AC C

367

EP

364

peroxidase and glutathione S-transferase from Exopalaemon carinicauda in response to Vibrio anguillarum and WSSV challenge, Fish Shellfish Immunol. 35 (2013) 661-670.

370

[7] Y.F. Duan, P. Liu, J.T. Li, Y. Wang, J. Li, P. Chen, Molecular responses of calreticulin gene to Vibrio

371

anguillarum and WSSV challenge in the ridgetail white prawn Exopalaemon carinicauda, Fish

372

Shellfish Immunol. 36 (2014) 164-171.

373

[8] I.E. Quintero-Reyes, K.D. Garcia-Orozco, R. Sugich-Miranda, A.A. Arvizu-Flores, E.F.

ACCEPTED MANUSCRIPT 374

Velazquez-Contreras, F.J. Castillo-Yañez, et al., Shrimp oncoprotein nm23 is a functional

375

nucleoside diphosphate kinase, J. Bioenerg Biomembr. 44 (2012) 325-331.

377

[9] L. Lascu, The nucleoside diphosphate kinases 1973-2000, J. Bioenerg Biomembr. 32 (2000) 213-214.

RI PT

376

[10] A.M. Gilles, E. Presecan, A. Vonica, I. Lascu, Nucleoside diphosphate kinase from human

379

erythrocytes Structural characterization of the two polypeptide chains responsible for heterogeneity

380

of the hexameric enzyme, J. Biol. Chem. 266 (1991) 8784-8789.

SC

378

[11] Y.N. Song, C.Y. Lu, J. Chen, G.F. Qiu, Characterization of a novel nm23 gene and its potential roles

382

in gametogenesis in the prawn Macrobrachium rosenbergii (de Man, 1879) (Crustacea: Decapoda),

383

Gene 531(1) (2013) 1-7.

M AN U

381

[12] X.K. Jin, W.W. Li, L. He, W. Lu, L.L. Chen, Y. Wang, et al., Molecular cloning, characterization and

385

expression analysis of two apoptosis genes, caspase and nm23, involved in the antibacterial

386

response in Chinese mitten crab, Eriocheir sinensis, Fish Shellfish Immunol. 30 (2011) 263-272.

387

[13] L. Amrein, P. Barraud, J.Y. Daniel, Y. Pérel, M. Landry, Expression patterns of nm23 genes during

390 391 392 393 394 395

EP

389

mouse organogenesis, Cell Tissue Res. 322 (2005) 365-378. [14] P.S. Steeg, G. Bevilacqua, L. Kopper, U.P. Thorgeirsson, J.E. Talmadge, L.A. Liotta, et al., Evidence

AC C

388

TE D

384

for a novel gene associated with low tumor metastatic potential, J. National Cancer Institute

80

(1988) 200-204.

[15] M.L. Lacombe, L. Milon, A. Munier, J.G. Mehus, D.O. Lambeth, The human Nm23/nucleoside diphosphate kinases, J. Bioenerg Biomembr. 32 (2000) 247-258. [16] Y.T. Tee, G.D. Chen, L.Y. Lin, J.L. Ko, P.H. Wang, Nm23-H1: a metastasisassociated gene, Taiwan J. Obstet Gynecol. 45 (2006) 107-113.

ACCEPTED MANUSCRIPT 396

[17] A.

Clavero-Salas,

R.R.

Sotelo-Mundo,

T.

Gollas-Galvan,

J.

HernandezLopez,

A.B.

Peregrino-Uriarte, A. Muhlia-Almazan, et al., Transcriptome analysis of gills from the white shrimp

398

Litopenaeus vannamei infected with White Spot Syndrome Virus, Fish Shellfish Immunol. 23 (2007)

399

459-472.

RI PT

397

[18] F. Yue, L.Q. Pan, P. Xie, D.B. Zheng, J. Li, Immune responses and expression of immune-related

401

genes in swimming crab Portunus trituberculatus exposed to elevated ambient ammonia-N stress,

402

Comp. Biochem. Physiol. A 157 (2010) 246-251.

SC

400

[19] W. Cheng, J.C. Chen, The virulence of Enterococcus to freshwater prawn Macrobrachium

404

rosenbergii and its immune resistance under ammonia stress, Fish Shellfish Immunol. 12 (2002)

405

97-109.

407

[20] J.C. Chen, Y.Z. Kou, Effects of ammonia on growth and molting of Penaeus japonicus juveniles, Aquaculture 104 (1992) 249-260.

TE D

406

M AN U

403

[21] J.C. Chen, C.Y. Lin, Oxygen consumption and ammonia-N excretion of Penaeus chinensis juveniles

409

exposed to ambient ammonia at different salinity levels, Comp. Biochem. Physiol. C 102 (1992)

410

287-291.

412 413

[22] J.E. Han, K.F.J. Tang, C.R. Pantoja, B.L. White, D.V. Lightner, qPCR assay for detecting and

AC C

411

EP

408

quantifying a virulence plasmid in acute hepatopancreatic necrosis disease (AHPND) due to pathogenic Vibrio parahaemolyticus, Aquaculture 442 (2015) 12-15.

414

[23] L. Tran, L. Nunan, R.M. Redman, L.L. Mohney, C.R. Pantoja, K. Fitzsimmons, et al.,

415

Determination of the infectious nature of the agent of acute hepatopancreatic necrosis syndrome

416

affecting penaeid shrimp, Dis. Aquat. Org. 105 (2013) 45-55.

417

[24] J. Joshi, J. Srisala, V.H. Truong, I.T. Chen, B. Nuangsaeng, O. Suthienkul, et al., Variation in

ACCEPTED MANUSCRIPT 418

Vibrio parahaemolyticus isolates from a single Thai shrimp farm experiencing an outbreak of

419

acute hepatopancreatic necrosis disease (AHPND), Aquaculture 428-429 (2014) 297-302. [25] Y.F. Duan, J.S. Zhang, H.B. Dong, Y. Wang, Q.S. Liu, H. Li, Oxidative stress response of the

421

black tiger shrimp Penaeus monodon to Vibrio parahaemolyticus challenge, Fish Shellfish

422

Immunol. 46 (2015) 354-365.

RI PT

420

[26] C.Z. Li, H.Y. Li, S. Wang, X. Song, Z.J. Zhang, Z. Qian, et al., The c-Fos and c-Jun from

424

Litopenaeus vannamei play opposite roles in Vibrio parahaemolyticus and white spot syndrome

425

virus infection, Dev. Comp. Immunol. 52 (2015) 26-36.

429 430 431 432

M AN U

428

marine decapods, Dev. Comp. Immunol. 7 (1983) 229-239.

[28] K. Tamura, J. Dudley, M. Nei, S. Kumar, MEGA 4: molecular evolutionary genetics analysis (MEGA) software version 4.0., Mol. Biol. Evol. 24 (2007) 1596-1599.

TE D

427

[27] K. Söderhäll, V.J. Smith, Separation of the haemocyte populations of Carcinus maenas and other

[29] S.V. Durand, D.V. Lightner, Quantitative real time PCR for the measurement of white spot syndrome virus in shrimp, J. Fish Dis. 25 (2002) 381-389. [30] J.P. Liang, J. Li, J.T. Li, P. Liu, F.Y. Dai, D.Y. Liu, Acute toxicity of ridgetail white prawn

EP

426

SC

423

Exopalaemon carinicauda, Fisheries Sci. 31(9) (2012) 526-529. [In Chinese]

434

[31] A.Y. Li, Seawater Chemistry, Beijing, China: China Agriculture Press (1995).

435

[32] K.J. Livak, T.D. Schmittgen, Analysis of relative gene expression data using realtime quantitative

436 437 438 439

AC C

433

PCR and the 2 (delta delta C(T)) method, Methods 25 (2001) 402-408.

[33] G. González-Rodríguez, A. Colubi, M.Á. Gil, Fuzzy data treated as functional data: a one-way ANOVA test approach, Comput Stat. Data Anal. 56 (2012) 943-955. [34] A.E. Toranzo, B. Magarinos, J.L. Romalde, A review of the main bacterial fish diseases in

ACCEPTED MANUSCRIPT 440

mariculture systems, Aquaculture 2005;246:37-61. [35] D.V. Lightner, K.W. Hasson, B.L. White, R.M. Redman, Experimental infections of western

442

hemisphere Penaeid shrimp with Asian white spot syndrome virus and Asian yellow head virus, J.

443

Aquat. Anim. Health 10 (1998) 271-281.

RI PT

441

[36] K.M. Spann, R.J.G. Lester, Special topic review: viral diseases of Penaeid shrimp with particular

445

reference to four viruses recently found in shrimp from Queensland, World J. Microbiol. Biotechnol.

446

13 (1997) 419-426.

SC

444

[37] W. Soonthornchai, W. Rungrassamee, N. Karoonuthaisiri, P. Jarayabhand, S. Klinbunga, K.

448

Söderhäll, et al., Expression of immune-related genes in the digestive organ of shrimp, Penaeus

449

monodon, after an oral infection by Vibrio harveyi, Dev. Comp. Immunol. 34 (2010) 19-28.

M AN U

447

[38] J. Du, H.X. Zhu, P. Liu, J. Chen, Y.J. Xiu, W. Yao, et al., Immune responses and gene expression

451

in hepatopancreas from Macrobrachium rosenbergii challenged by a novel pathogen spiroplasma

452

MR-1008, Fish Shellfish Immunol. 34 (2013) 315-323.

455 456

(2007) 697-743.

EP

454

[39] B. Lemaitre, J. Hoffmann, The host defense of Drosophila melanogaster, Annu. Rev. Immunol. 25

[40] S. Iwanaga, B.L. Lee, Recent advances in the innate immunity of invertebrate animals, J. Biochem.

AC C

453

TE D

450

Mol. Biol. 38 (2005) 128-150.

457

[41] P. Chen, J.T. Li, B.Q. Gao, P. Liu, Q.Y. Wang, J. Li, cDNA cloning and characterization of

458

peroxiredoxin gene from the swimming crab Portunus trituberculatus, Aquaculture 322-323 (2011)

459

10-15.

460

[42] J.X. Tian, J. Chen, T. Jiang, S.A. Liao, A.L. Wang, Transcriptional regulation of extracellular copper

461

zinc superoxide dismutase from white shrimp Litopenaeus vannamei following Vibrio alginolyticus

ACCEPTED MANUSCRIPT

465 466 467 468 469

J. Invertebr. Pathol. 106 (2011) 110-130. [44] G. Aguirre, F. Ascencio, Infectious disease in shrimp species with aquaculture potential, Rec. Res.

RI PT

464

[43] D.V. Lightner, Virus diseases of farmed shrimp in the western hemisphere (the Americas): a review,

Dev. Microbiol. 4 (2000) 333-348.

[45] A. Bilitou, J. Watson, A. Gartner, S. Ohnuma, The NM23 family in development, Mol. Cell Biochem. 329 (2009) 17-33.

SC

463

and WSSV infection, Fish Shellfish Immunol. 30 (2007) 234-240.

[46] R.B. Pyles, R.L. Thompson, Mutations in accessory DNA replicating functions alter the relative

M AN U

462

470

mutation frequency of herpes simplex virus type 1 strains in cultured murine cells, J. Virol. 68 (1994)

471

4514-4524.

[47] H.F. Tzeng, Z.F. Chang, S.E. Peng, C.H. Wang, J.Y. Lin, G.H. Kou, et al., Chimeric polypeptide of

473

thymidine kinase and thymidylate kinase of shrimp white spot syndrome virus: thymidine kinase

474

activity of the recombinant protein expressed in a baculovirus/insect cell system, Virol. 299(2)

475

(2002) 248-255.

TE D

472

[48] J.H. Leu, S.H. Chen, Y.B. Wang, Y.C. Chen, S.Y. Su, C.Y. Lin, et al., A review of the major penaeid

477

shrimp EST studies and the construction of a shrimp transcriptome database based on the ESTs from

AC C

478

EP

476

four penaeid shrimp, Marine Biotech. 13(4) (2011) 608-621.

479

[49] Y.Y. Chen, S.S. Sim, S.L. Chiew, S.T. Yeh, C.H. Liou, J.C. Chen, Dietary administration of a

480

Gracilaria tenuistipitata extract produces protective immunity of white shrimp Litopenaeus

481

vannamei in response to ammonia stress, Aquaculture 370 (2012) 26-31.

482

[50] W. Cheng, J.C. Chen, The virulence of Enterococcus to freshwater prawn Macrobrachium

483

rosenbergii and its immune resistance under ammonia stress, Fish Shellfish Immunol. 12 (2002)

ACCEPTED MANUSCRIPT 484 485 486

97-109. [51] G. Le Moullac, P. Haffner, Environmental factors affecting immune responses in Crustacea, Aquaculture 191 (2000) 121-131. [52] Q.Q. Ge, J.P. Liang, J.T. Li, J. Li, Y.F. Duan, F.Z. Zhao, et al., Molecular cloning and expression

488

analysis of Relish gene from the ridgetail white prawn Exopalaemon carinicauda, Fish Sci. 81

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(2015) 699-711.

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Fig. 1. Nucleotide and deduced amino acid sequences of EcNM23 cDNA of E. carinicauda. The letters

507

in box indicated the start codon (ATG) and the polyadenylation signal sequence (AATAA). Asterisk

508

indicated the stop codon (TAA). The NM23 family signature motifs (NIIRGSDSI) were underlined.

RI PT

509 Fig. 2. Multiple alignment of EcNM23 with other known NM23s: NM23 of M. rosenbergii

511

(AGO05949), L. vannamei (ABI93176), P. monodon (AFL02665), E. sinensis (ADK94169), I.

512

punctatus (NP_001187018), C. mydas (XP_007062842) and G. gallus (NP_990378); NM23-H1

513

(NP_937818) and NM23-H2 (NP_001018149) of H. sapiens. The NM23 signature motifs was marked

514

by frame.

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Fig. 3. Phylogenetic tree of different species NM23s on the basis of the amino acid sequence using

517

neighbor-joining distance analysis. The protein sequences used for phylogenetic analysis were as

518

follows: M. rosenbergii NM23 (AGO05949); L. vannamei NM23 (ABI93176); P. monodon NM23

519

(AFL02665); E. sinensis NM23 (ADK94169); I. punctatus NM23 (NP_001187018); D. rerio

520

NM23-H1 (AAF20910); H. sapiens: NM23-H1 (NP_937818), NM23-H2 (NP_001018149), NM23-H3

521

(NP_002504), NM23-H4 (NP_005000), NM23-H5 (NP_003542), NM23-H6 (NP_005784), NM23-H7

522

(NP_037462), NM23-H8 (NP_057700), NM23-H9 (NP_835231), NM23-H10 (NP_008846); Mus

523

musculus: NM23-H1 (NP_032730), NM23-H2 (NP_032731), NM23-H3 (NP_062704), NM23-H4

524

(NP_062705), NM23-H5 (NP_542368), NM23-H6 (NP_061227), NM23-H7 (NP_612187), NM23-H8

525

(NP_853622), NM23-H9 (XP_893103), NM23-H10 (NP_598430). The numbers at the forks indicated

526

the bootstrap.

527

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ACCEPTED MANUSCRIPT 528

Fig. 4. Tissue specific expression of EcNM23 mRNA related to hepatopancreas expression by the

529

real-time PCR. The reference gene is 18S rRNA. Vertical bars represent the mean ± SD (N = 3).

530 Fig. 5. Cumulative survival of E. carinicauda at different time after V. parahaemolyticus and WSSV

532

challenge and ammonia-N stress treatment. VP: V. parahaemolyticus challenge, WSSV: WSSV

533

challenge, ammonia-N: ammonia-N stress.

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Fig. 6. The mRNA expression levels of EcNM23 in hemocytes (a) and hepatopancreas (b) of E.

536

carinicauda at different time intervals after V. parahaemolyticus challenge treatment. The reference

537

gene is 18S rRNA. Vertical bars represented the mean ± SD (N = 3). Significant differences (P < 0.05)

538

of EcNM23 expression between the challenged and the control group were indicated with asterisks.

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Fig. 7. The mRNA expression levels of EcNM23 in hemocytes (a) and hepatopancreas (b) of E.

541

carinicauda at different time intervals after WSSV challenge treatment. The reference gene is 18S

542

rRNA. Vertical bars represented the mean ± SD (N = 3). Significant differences (P < 0.05) of EcNM23

543

expression between the challenged and the control group were indicated with asterisks.

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Fig. 8. The mRNA expression levels of EcNM23 in hepatopancreas (a) and gills (b) of E. carinicauda

546

at different time intervals after ammonia-N stress. The reference gene is 18S rRNA. Vertical bars

547

represented the mean ± SD (N = 3). Significant differences (P < 0.05) of EcNM23 expression between

548

the challenged and the control group were indicated with asterisks.

ACCEPTED MANUSCRIPT Table 1 Primer sequences used in this study. Primer name

Sequence (5'-3')

RI PT

EcNM23 GAACGCACTTTCATCGCCGT

R1 (reverse)

ACAGCTACACAGTCAGTCCT

F2 (forward)

CCTTTCTACCCAGGACTTTGC

R2 (reverse)

CCATCATTACACGGGCTGTT

UPM

CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT

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F1 (forward)

CTAATACGACTCACTATAGGGC 18S rRNA

TATACGCTAGTGGAGCTGGAA

18S-HR

GGGGAGGTAGTGACGAAAAAT

F3 (forward)

TGCCTTGCCGGAAATTAGTG

AC C

R3 (reverse)

ACAATGGTCCCGTCCTCATC

EP

WSSV

TE D

18S-HF

Probe (T)

TET-CAGAAGCCATGAAGAATGCCGTCTATCAC-TAMRA

AC C

EP

TE D

M AN U

SC

RI PT

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AC C

EP

TE D

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RI PT

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AC C

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TE D

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SC

RI PT

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TE D

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AC C

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TE D

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SC

RI PT

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AC C

EP

TE D

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SC

RI PT

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AC C

EP

TE D

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SC

RI PT

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AC C

EP

TE D

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SC

RI PT

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AC C

EP

TE D

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AC C

EP

TE D

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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT A oncoprotein NM23 (EcNM23) was cloned and characterized from Exopalaemon carinicauda; The expression of EcNM23 in various tissues was investigated; expression

profiles

were

evaluated

after

pathogens

(Vibrio

RI PT

EcNM23

AC C

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SC

parahaemolyticus and WSSV) challenge and ammonia-N stress.