A class B scavenger receptor from Eriocheir sinensis (EsSR-B1) restricts bacteria proliferation by promoting phagocytosis

Accepted Manuscript A class B scavenger receptor from Eriocheir sinensis (EsSR-B1) restricts bacteria proliferation by promoting phagocytosis Yao-Meng Wu, Lei Yang, Xue-Jie Li, Lu Li, Qun Wang, Wei-Wei Li PII:

S1050-4648(17)30553-3

DOI:

10.1016/j.fsi.2017.09.034

Reference:

YFSIM 4824

To appear in:

Fish and Shellfish Immunology

Received Date: 12 June 2017 Revised Date:

4 September 2017

Accepted Date: 9 September 2017

Please cite this article as: Wu Y-M, Yang L, Li X-J, Li L, Wang Q, Li W-W, A class B scavenger receptor from Eriocheir sinensis (EsSR-B1) restricts bacteria proliferation by promoting phagocytosis, Fish and Shellfish Immunology (2017), doi: 10.1016/j.fsi.2017.09.034. 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

A class B scavenger receptor from Eriocheir sinensis (EsSR-B1)

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restricts bacteria proliferation by promoting phagocytosis

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Yao-Meng Wu1, Lei Yang1, Xue-Jie Li, Lu Li, Qun Wang*, Wei-Wei Li*

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4 Laboratory of Invertebrate Immunological Defense & Reproductive Biology, School

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of Life Science, East China Normal University, Shanghai, China

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*Corresponding authors:

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Wei-Wei Li, [email protected], Laboratory of Invertebrate Immunological

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Defense & Reproductive Biology, School of Life Science, East China Normal

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University, Shanghai, China.

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Qun Wang, [email protected], Laboratory of Invertebrate Immunological

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Defense & Reproductive Biology, School of Life Science, East China Normal

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University, Shanghai, China. 1

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ABSTRACT

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Scavenger receptors (SRs) are important pattern recognition receptors (PRRs), which

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play significant roles

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pathogen-associated molecular patterns (PAMPs). In this study, we report the cloning

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and characterization of a SR from Eriocheir sinensis (EsSR-B1) which is a 500 amino

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acid protein encoded by a gene comprised of 2726 nucleotides with a 1503 bp open

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reading frame. The domains of EsSR-B1 were found to be evolutionarily conserved.

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EsSR-B1 was widely detected in different tissues of E. sinensis and significantly

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up-regulated in hemocytes after stimulation by Staphyloccocus aureus or Vibrio

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parahaemolyticus. Recombinant EsSR-B1 protein could bind to bacteria and promote

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phagocytosis upon bacterial stimulation. Moreover, antimicrobial peptide expression

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was reduced in EsSR-B1-silenced hemocytes after challenge by S. aureus or V.

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parahaemolyticus. Thus, EsSR-B1 has a critical role in the binding of bacteria and

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subsequent promotion of hemocyte phagocytosis.

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in host defense against pathogens by identifying

Keywords: Eriocheir sinensis, EsSR-B1, phagocytosis, antimicrobial peptide

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1. Introduction Phagocytosis is the cornerstone of innate and acquired immune responses to

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pathogens [1] and highly related to immune surveillance, embryonic development and

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metabolic homeostasis, which is an evolutionarily conserved process [2, 3].

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Invertebrates have an unique modality, so-called innate immunity, to detect and

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respond

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lipopolysaccharides (LPS), lipoteichoic acids, lipoproteins, peptidoglycan (PGN) and

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β-1,3-glucans [4, 5]. Among the major host defense systems, the phagocytic system

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cooperates with other immune reactions to protect the host from invading pathogens

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[4, 6]. During phagocytosis, various pattern recognition receptors (PRRs) are

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expressed on the cell surface to recognize conserved molecular motifs of pathogenic

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microorganisms [7, 8]. Different types of PRRs, such as Toll/Toll-like receptors

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(TLRs), Nod-like receptors (NLRs), RIG-like receptors (RLRs) and scavenger

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receptors (SRs) [9, 10], can bind to invading pathogens to activate numerous signaling

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pathways. However, relatively few related studies have been reported in invertebrates,

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and the role of SRs in phagocytosis is still unclear.

molecular

patterns

(PAMPs),

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pathogen-associated

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to

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The concept of SRs was first described by Brown and Goldstein in the 1970s [11].

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SRs form a large family of transmembrane cell surface glycoproteins that include an

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extensive range of molecules involved in receptor-mediated endocytosis of selected

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polyanionic ligands, including modified low-density lipoproteins (LDL) [12, 13].

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More recently, SRs were divided into ten different classes (A–J) according to their 3

ACCEPTED MANUSCRIPT structure and biological function [14]. Moreover, several reports had demonstrated the

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significant role of SRs in innate immune defense by acting as PRRs [15]. SRs have

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two functions in cells. Some SRs were indicated to have a potential role in host

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defense as PRRs by binding to different types of pathogens [16], while other SRs

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were found to be able to clear apoptotic cells and cellular debris by recognizing

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damage-associated molecular patterns (DAMPs). Recent studies also demonstrated

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some SRs as co-receptors for TLRs, especially TLR2, in pro-inflammatory cytokine

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responses to various PAMPs [17, 18]. Interestingly, several pathogens apparently have

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evolved mechanisms to escape recognition by SRs [19, 20].

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Class B SRs (SR-Bs) are the most widely studied among SRs interacting with

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pathogenic microorganisms [21]. SR-Bs include receptors recently known as SR-BI,

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SR-BII, CD36 and LIMP2 in mammals [22]. These four members have two

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transmembrane domains with both the amino and carboxyl termini located within the

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cytoplasm, which straddle a central domain of 400~450 residues that is glycosylated

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and mediates ligand recognition [23]. As the first SR-B to be cloned and molecularly

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characterized, CD36 was found to be mainly distributed in insulin-responsive cells

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and hematopoietic cells such as platelets, monocytes and macrophages. According to

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previous studies, CD36 was known to be involved in migration, signaling and

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inflammatory processes [22]. In addition, CD36 plays an important role in innate

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immunity against bacteria, especially Gram-positive pathogens [24].

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ACCEPTED MANUSCRIPT SR-Bs had been identified in several invertebrates, such as Drosophila

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melanogaster, Anopheles gambiae and Marsupenaeus japonicas [25, 26]. Recently, a

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novel member of the SR-B family in kuruma shrimp M. japonicas, called MjSR-B1,

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was reported. It is a phagocytic receptor which can bind to both Gram-positive and

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Gram-negative bacteria and regulate expression of antimicrobial peptides (AMPs)

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[25]. In addition, Croquemort (SCRBQ) is a type of SR-B and a member of the CD36

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family [27]. A novel SCRBQ from kuruma shrimp M. japonicus called MjSCRBQ

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was found to be widely expressed in all tissues and at high levels in the brain [28].

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However, few studies of SRs in crabs have been reported.

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Eriocheir sinensis, one of the most vital aquaculture crustacean species in

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southeast Asia [29], is threatened by many outbreaks of diseases that result in decline

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of production and economic loss [30]. Hydroperitoneum disease and trembling

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disease are two kinds of bacteria diseases mainly happening in Eriocheir sinensis,

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which are caused by Aeromonas hydrophlia and Vibrio mimicus [31]. In this study, we

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obtained a SR-B cDNA from E. sinensis and designated it as EsSR-B1. Its expression

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was upregulated after challenge with Gram-positive and Gram-negative bacteria.

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Furthermore, the recombinant EsSR-B1 protein (rEsSR-B1) was expressed and

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purified. In vitro experiments indicated that EsSR-B1 could bind to and agglutinate

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both Gram-positive bacteria and Gram-negative bacteria. RNA interference of

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EsSR-B1 reduced the phagocytosis rate of crab hemocytes and decreased the

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expression level of AMP genes. Therefore, EsSR-B1 is a phagocytic receptor against

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Gram-positive bacteria and Gram-negative bacteria and can regulate expression of

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AMPs.

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2. Materials and methods

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2.1. Animals

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Healthy adult Chinese mitten crabs (n = 180; 100 ± 15 g wet weight) were

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purchased from the Xin’An Market in Shanghai, China and maintained in filtered,

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aerated freshwater for a week under constant temperature (20-25°C) before use in

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experiments every day.

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2.2. Bacterial challenge and sample collection

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Two bacteria species Vibrio parahaemolyticus (BYK00036) and Staphyloccocus

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aureus (BYK0113) were obtained from the National Pathogen Collection Center for

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Aquatic Animals (Shanghai Ocean University, Shanghai, China). After overnight

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culture in Luria-Bertani medium, the bacteria were collected by centrifugation at 5000

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× g for 3 min, washed three times with sterile phosphate-buffered saline (PBS; 137

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mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 2 mM KH2PO4, pH 7.4), resuspended

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in PBS and plated for colony counting. For bacterial challenge, 180 crabs were

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random divided into three groups (sex ratio, 1:1). After adjusting the bacteria to 1 ×

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106 CFU/ml, the bacterial suspension (100 ml) was injected into each crab in the first

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group (S. aureus challenge) and second group (V. parahaemolyticus challenge). The

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control groups were injected with 100 ml PBS. More than five crabs in each group

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ACCEPTED MANUSCRIPT were selected randomly at specific time intervals [0 h (blank control), 12 h, 24 h, 36 h

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and 48 h] after bacterial challenge. Hemolymph was separated from the hemocoel in

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the arthrodial membrane of the last pair of walking legs of each crab using a syringe

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(approximately 5 ml per crab) with an equal volume of anticoagulant solution (0.1 M

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glucose, 30 mM citrate, 26 mM citric acid, 0.14 M NaCl, 10 mM EDTA) [32].

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Thereafter, hemocytes were obtained by centrifuging (800 × g at 4 °C, 10 min) the

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collected hemolymph and stored at -80°C. Other tissues, including heart,

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hepatopancreas, stomach, gills, muscles and intestine, were extracted from the control

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group crabs and stored at -80°C for cloning and expression analysis. Primary

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hemocytes of E. sinensis were cultured and collected according to the previous study

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of our laboratory [33].

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2.3. Total RNA extraction and first-strand cDNA synthesis

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According to the manufacturer's protocol, total RNA was extracted from E.

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sinensis by using the Trizol Reagent (Invitrogen, Carlsbad, CA, USA). The

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concentration and quality of the total RNA were evaluated and analyzed by 1%

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agarose-gel electrophoresis (AGE) and the NanoDrop 2000 spectrophotometer

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(Thermo Fisher Scientific, USA).

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For full-length cDNA cloning, total RNA (5 µg) was reverse transcribed using the

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SMARTerTM RACE cDNA Amplification kit (Clontech, Shiga, Japan). For

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quantitative real-time RT-PCR (qRT-PCR) analysis, total RNA (4 µg) was reverse

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transcribed using the PrimeScript™ Real-time PCR Kit (TaKaRa, Shiga, Japan) 7

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according to the manufacturer’s protocols.

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2.4. Gene cloning of EsSR-B1 A partial cDNA sequence [expressed sequence tag (EST)] of EsSR-B1 was

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obtained from the hemocyte cDNA library (unpublished) of the Chinese mitten crab.

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Partial EsSR-B1 cDNA sequences were extended using 5’ and 3’ RACE (SMARTer®

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RACE cDNA Amplification kit, Clontech, Japan) under the instructions of

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manufacturer, and gene-specific primers (Table 1) were designed based on the original

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EST sequence. The PCR procedure was as follows: 94°C for 4 min; 35 cycles at 94°C

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for 30 s, 62°C for 30 s, and 72°C for 1 min 30 s; 72°C for 10 min. Finally, the

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products of PCR and RACE-PCR were purified and inserted into the pEASY-T1

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vector (TransGen, Beijing, China) for sequencing. The whole nucleotide sequence

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was submitted to GenBank with the accession number MF055657.

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2.5. Bioinformatic analysis

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The obtained sequence was compared against sequences from other representative

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vertebrates and invertebrates in the NCBI database using the online Search Tool

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(BLASTX) (http://www.ncbi.nlm.nih.gov/). The open reading frame (ORF) was

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analyzed

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(http://www.ncbi.nlm.nih.gov/gorf/orfig.cgi). The structure and functional domain of

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EsSR-B1 was predicted by both ExPASy (http://prosite.expasy.org/prosite.html/) and

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SMART

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(http://www.cbs.dtu.dk/services/TMHMM/) was used to predict transmembrane

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and

identified

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(http://smart.embl-heidelberg.de/).

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the

TMHMM

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finder

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using the online software SWISS-MODEL (http://swissmodel.expasy.org/). The

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ClustalX2.0 program and DNAMAN software were used to perform multiple

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sequence alignment. Furthermore, to detect the evolutionary relationships of EsSR-B1,

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a phylogenetic tree was constructed with EsSR-B1 and other 24 related proteins by

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using the neighbor-joining (NJ) method of MEGA6 software with 1000 replications.

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2.6. qRT-PCR analysis of EsSR-B1

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Specific primers for EsSR-B1 (SR-B1 qF and SR-B1 qR, Table 1) and β-actin

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[34-36] (β-actin qF and β-actin qR, Table 1) were used to amplify the cDNA

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fragments. Expression of EsSR-B1 in different tissues was analyzed by qRT-PCR

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using SYBR® Premix Ex TaqTM (Tli RNaseH Plus) (TaKaRa) and CFX96 Real-Time

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System (Bio-Rad, Hercules, CA, USA). The reaction conditions were as follows:

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95°C for 2 min; 39 cycles at 95°C for 5 s and 58°C for 30 s. The gene expression data

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were analyzed using CFX Manager™ software, and relative expression levels were

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calculated and quantified using the comparative CT method (2−∆∆Ct method) [37].

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Data from three independent replications were analyzed by the least square difference

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(LSD) method, and the significance was set at P < 0.05. The data were assessed by a

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t-test using IBM SPSS Statistics 20 software. For expression of EsSR-B1 after

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bacterial challenge, the same primers and reaction conditions were used as detailed in

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this section.

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2.7. Recombinant expression and purification of the extracellular region of

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EsSR-B1 The primers (EsSR-B1 YF and EsSR-B1 YR, Table 1) were designed to amplify

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the complete extracellular region of EsSR-B1 by Primer Premier 5.0 software. The

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products were purified, sequenced by Invitrogen and inserted into the pEASY-T1

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vector (TransGen, Beijing, China). The constructed pEASY-T1-SRB1 plasmid and

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pET28a (Novagen, Darmstadt, Germany) plasmid were double digested with

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endonucleases EcoRI and XhoI (Takara). Next, restricted DNA products were ligated

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using T4 DNA ligase (NewEngland Biolabs, USA) at 16°C overnight to construct the

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recombinant expression vector pET28a-SRB1. The recombinant vector was

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sequenced by Invitrogen and checked by EcoRI and XhoI enzyme digestion and PCR.

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The recombinant expression plasmid pET28a-SRB1 was extracted and

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transformed into Escherichia coli Transetta (DE3) host cells from TransGen (Beijing,

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China). Single colonies of the transformants were picked up and used for the

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expression of rEsSR-B1 protein when the OD600 value reached 0.6 (the logarithmic

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phase). In addition, two conditions were established to induce the rEsSR-B1 protein

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expression: 0.25 mM Isopropyl-b-D-thio-galactoside (IPTG) for 3 h at 37°C and 1

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mM IPTG for 3 h at 37°C with 200 rpm shaking. The bacteria were collected by

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centrifugation (6000 × g, 5min) and washed with cold PBS repeatedly, and sonicated

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(40 W-60 W) on ice until the sample was clear. The cell lysate was collected and

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resuspended in binding buffer (20 mM Tris-HCl, 50 mM NaCl, 20 mM imidazole and

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8 M urea). The sample was loaded onto a Ni-NTA His-TrapTM-FF crude column (GE

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ACCEPTED MANUSCRIPT Healthcare). The resuspension solution was washed with imidazole elution buffer [20

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mM Tris-Hcl, 50 mM NaCl, x mM imidazole (x=40, 60, 80, 100, 200, 300, 500)] at 1

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ml/min. Washing solutions were collected and examined by sodium dodecyl sulfate

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polyacrylamide gel electrophoresis (SDS-PAGE) on a 12% gel. The rEsSR-B1 protein

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was refolded by using graded urea dialysis. Purified rEsSR-B1 was placed into a

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dialysis bag, which was immersed in a 2 L beaker containing different concentrations

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of graded urea buffer solution (6 M, 4 M, 2 M, 1 M, 0 M urea, 20 mM Tris-HCL, 50

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mM NaCl) for 12 h at 4°C with stirring. The renatured rEsSR-B1 was then analyzed

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by 12% SDS-PAGE.

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2.8. Bacteria binding activities and Western blotting

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Three Gram-positive bacteria (S. aureus, Bacillus subtilis and Microbacterium

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lactium) and three Gram-negative bacteria (V. parahaemolyticus, Aeromonas

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hydrophila and E. coli) were cultured overnight in liquid medium (LB for bacteria).

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The bacteria were collected by centrifugation at 6000 × g and resuspended in 2 ml 1×

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TBS (Cwbio, Shanghai, China). Thereafter, the bacteria (2 × 107 CFU/ml in 500 ml of

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TBS) were mixed and incubated with 200 µl renatured rEsSR-B1 (0.5 mg/ml) (section

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2.7) for 1 h at 37°C. After centrifugation at 6000 × g for 5 min, the bacteria were

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collected once again, washed with TBS three times and finally resuspended in TBS.

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SDS-PAGE loading buffer was added to each sample and heated at 100°C for 8 min.

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The samples were then separated by 12% SDS-PAGE and electrophoretically

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transferred to a polyvinylidene fluoride (PVDF) membrane (Cwbio). The membrane

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TBS containing 0.1% Tween-20) at room temperature on a shaker for 1 h and

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incubated with an anti-His-tag mouse antibody (1:2000) (Cwbio) diluted with TBST

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at 4°C overnight. The membrane was washed several times with TBST for 6 min and

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then incubated with horseradish peroxidase-conjugated goat anti-mouse IgG antibody

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(1:3000) (Cwbio) for 1 h at 37°C. After washing several times with TBST, the

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immunoreactive protein bands were visualized using the ChemiDoc XRS imaging

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system (Bio-Rad).

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2.9. RNA interference (RNAi) in vitro

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For the RNAi experiment, the siRNAs of EsSR-B1 and GFP (as control) were

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obtained from GenePharma (Shanghai, China), and the primer sequences are listed in

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Table 1. Five micrograms of EsSR-B1 or GFP siRNA was transfected into cultured

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primary hemocytes of E. sinensis for 24 h using the siRNA-Mate reagent

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(GenePharma). A total of 3 × 106 hemocytes were in each 60 mm dish (Corning,

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Corning, NY, USA) in a volume of 4 ml. Sterile PBS was added to the controls. The

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method for culturing primary E. sinensis hemocytes is described in Section 2.2. The

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transfection protocol was carried out according to the manufacturer’s instructions.

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Total RNA was extracted to detect the RNAi efficiency.

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2.10. Expression of AMP genes after RNAi of EsSR-B1

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After RNAi of EsSR-B1, the E. sinensis hemocytes were challenged by S. aureus

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or V. parahaemolyticus. The hemocytes were collected to extract RNA 12 h after 12

ACCEPTED MANUSCRIPT bacterial challenge. qRT-PCR was performed to test the expression level of AMP

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genes. The AMPs were EsDWD1 [38] (DWD1 qF and DWD1 qR), Lys [39] (Lys qF

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and Lys qR), EsALF1 [40] (ALF1 qF and ALF1 qR), EsALF2 [41] (ALF2 qF and

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ALF2 qR), EsALF3 [42] (ALF3 qF and ALF3 qR), CrusEs1 [43] (Crus-1 qF and

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Crus-1 qR) and CrusEs2 [44] (Crus-2 qF and Crus-2 qR). All primers for AMPs are

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shown in Table 1, and all experiments were performed in triplicate.

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2.11. Fluorescent labeling of bacteria and phagocytosis assay

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The first step of labeling V. parahaemolyticus and S. aureus with fluorescent

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isothiocyanate (FITC) (Sigma, St. Louis, MO, USA) was conducted as previously

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described. After culture, the collected bacteria were washed twice with PBS, heated at

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70°C for 30 min, washed with 0.1 M NaHCO3 and then incubated in 0.1 M NaHCO3

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containing 0.1 mg/ml FITC for 1 h at room temperature. Subsequently, the

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FITC-labeled bacteria were rinsed with PBS until no dissociated FITC was visible.

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The E. sinensis hemocytes were cultured as previously described in Section 2.2

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with slight modifications for seeding onto a cover glass according to Wang et al. [45].

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After RNAi of EsSR-B1 or GFP, each dish was instilled with 10 µl FITC-labeled

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bacteria (1 × 109 CFU/ml). The phagocytosis assay was performed as follows. At 1.5

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h after bacterial challenge, the hemocytes mounted on slides were washed twice with

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PBS (4°C), fixed in 4% paraformaldehyde for 10 min and washed thoroughly with

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PBS (4°C) at least three times. The slides were then stained with a nuclear

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counterstain DAPI (blue), quenched with 0.4% Typan Blue (Sigma) and finally

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observed under a fluorescence microscope (Leica, Germany). The remaining cultured hemocytes were washed twice with PBS (4°C), and 0.2 ml

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0.25% trypsin-EDTA (Gibco, Carlsbad, CA, USA) was added in each dish for 2 min.

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The collected hemocytes were then used to examine the phagocytosis ratio by flow

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cytometry using the CytoFLEX instrument (Beckman Coulter). After drawing the gate,

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the phagocytic rate was displayed in the upper right corner of the image. The assay

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was performed three times, and data were analyzed using CytExpert software.

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2.12. Statistical analysis

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SPSS software (Ver11.0) was used for statistical analysis. Statistical significance

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was calculated by the least square difference (LSD) method. Significance was set at P

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< 0.05.

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3. Results

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3.1. Characterization of full-length EsSR-B1 cDNA

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The cloned full-length EsSR-B1 cDNA (GenBank accession number MF055657)

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contains 2726 bp, with a 1503 bp ORF encoding a 500 amino acid protein, a 619 bp 5'

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untranslated region (UTR) and a 604 bp 3' UTR (Fig. 1). The theoretical molecular

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mass of EsSR-B1 is 56.66 kDa, and its isoelectric point is 8.55. EsSR-B1 was

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determined to have a CD36 domain (aa 12-466) that contains two transmembrane

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regions at the N-terminal (aa 7-29) and C-terminal (aa 442-464) of the protein by

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using the SMART program (Fig. 2A) and the TMHMM Server (Fig. 2B). BLASTX

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ACCEPTED MANUSCRIPT analysis showed that the EsSR-B1 has 63% identity with the SR-B1 of M. japonicus

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and 35% identity with the CD36-like protein of Branchiostoma japonicum. A

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three-dimensional model of EsSR-B1 was constructed by using the online software

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SWISS-MODEL (Fig. 2C). The multiple alignment results showed relatively low

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conservation of the SR-B family (Fig. 3).

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We found some SR-As, SR-Bs and SR-Cs from GenBank for phylogenetic

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analysis using protein sequences. As might be expected, the selected SRs were

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divided into three groups, SR-A, SR-B and SRC. The SR-Bs were divided into two

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subgroups, and EsSR-B1 was found to belong to subgroup I (Fig. 4).

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3.2. Tissue expression pattern of EsSR-B1

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qRT-PCR was used to analyze the tissue distribution and expression pattern of

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EsSR-B1. The results showed that EsSR-B1 expression was widely observed in all the

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detected tissues of E. sinensis, and it was abundant in immune-related tissues such as

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the heart, gills, intestine and hepatopancreas (Fig. 5).

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3.3. Temporal expression of EsSR-B1 in hemocytes after bacterial challenge

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S. aureus and V. parahaemolyticus were used to respectively represent

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Gram-negative bacteria and Gram-positive bacteria [46]. The expression profiles of

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EsSR-B1 in hemocytes after injection with the two bacteria species were analyzed by

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qRT-PCR. In response to S. aureus challenge, the expression level of EsSR-B1 was

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upregulated at 24 h, 36 h and 48 h, above the control (Fig. 6A). After V.

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parahaemolyticus challenge, compared with the control, the expression level of 15

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EsSR-B1 was also upregulated from 12 h to 48 h, especially, at the 24 h and 48 h time

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points (Fig. 6B).

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3.4. Expression, purification and renaturation of rEsSR-B1 The extracellular region of EsSR-B1 was expressed in E. coli Rosetta with the

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pET28a system. Since the recombinant protein consists of the extracellular region of

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EsSR-B1 plus a His-tag, the molecular mass of rEsSR-B1 was predicted to be

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approximately 52 kDa, and the PI was predicted to be 6.41. In contrast to non-induced

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Transetta (Fig. 7, lane 1), a thick band (Fig. 7, lane 2) was observed at the molecular

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weight of 52 kDa, which was consistent with the predicted rEsSR-B1 molecular mass

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and showed that rEsSR-B1 was expressed successfully. The purification of the

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recombinant protein was achieved at an optimal concentration of imidazole (300 mM),

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which yielded highly pure rEsSR-B1 (Fig. 7, lane 3). Through stepwise dialyses,

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rEsSR-B1 was successfully obtained and dissolved in PBS (Fig. 7, lane 4).

328

3.5. EsSR-B1 binding to bacteria

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A binding assay was designed to determine the ability of rEsSR-B1 to bind

330

Gram-negative bacteria (E. coli, A. hydrophila, V. parahaemolyticus) and

331

Gram-positive bacteria (S. aureus, B. subtilis, M. lactium). In vitro binding assays,

332

rEsSR-B1 could bind to all of the tested bacteria with different levels of affinity.

333

Meanwhile, the ability of rEsSR-B1 to bind E. coli and M. lactium was weaker than

334

that of other tested bacteria (Fig. 8).

335

3.6. EsSR-B1 mediates phagocytosis in E. sinensis

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ACCEPTED MANUSCRIPT To investigate the phagocytosis of EsSR-B1 in vitro, RNAi of EsSR-B1 and

337

fluorescent labeling of bacteria were performed. qRT-PCR analysis indicated that 24 h

338

after transfection of siEsSR-B1 and siGFP, the mRNA level of EsSR-B1 in hemocytes

339

declined significantly in contrast to the control (Fig. 9A). The results showed that

340

RNAi of EsSR-B1 significantly decreased the phagocytic rate for Gram-positive and

341

Gram-negative bacteria (Fig. 9B). The phagocytosis rate was calculated to decrease

342

from approximately 15% to 7% for Gram-positive bacteria and from approximately

343

21% to 15% for Gram-negative bacteria (Fig. 9C). Also, the data also showed that

344

RNAi of EsSR-B1 reduced the phagocytotic rate by using flow cytometry (Fig. 9D).

345

These results demonstrated that EsSR-B1 promoted phagocytosis of both

346

Gram-positive and Gram-negative bacteria in E. sinensis.

347

3.7. Expression of antimicrobial peptides is mediated by EsSR-B1

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We next investigated whether EsSR-B1 participates in restricting the bacteria

349

proliferation by regulating the expression of antimicrobial peptides (AMPs), including

350

double WAP domain-containing protein (EsDWD1), lysozyme (EsLys), crustins

351

(CrusEs1, CrusEs2) and antilipopolysaccharide factors (EsALF1, EsALF2 and

352

EsALF3). Therefore, the expression of AMPs was analyzed by qRT-PCR in

353

EsSR-B1-silenced hemocytes of E. sinensis challenged by S. aureus or V.

354

parahaemolyticus. The results showed that EsDWD1, EsLys and CrusEs1 were

355

involved in the defense against Gram-positive bacteria based on the decrease in their

356

expression levels after EsSR-B1 silencing (Fig. 10A). EsDWD1, EsALF1, EsALF2,

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ACCEPTED MANUSCRIPT 357

EsALF3 and CrusEs2 were involved in anti-Gram-negative bacterial responses (Fig.

358

10B).

359

4. Discussion

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In this study, a novel SR-B (EsSR-B1) with remarkable phagocytic activity is

362

cloned and identified from E. sinensis. This is the first report in Chinese mitten crab

363

of the identification and characterization of a SR-B. Class B SRs are type III

364

transmembrane receptors with two transmembrane domains, an extracellular loop

365

with multiple glycosylation sites and two short intracellular tails. The extracellular

366

loop has a plurality of specific ligand binding sites, thus allowing it to mediate antigen

367

recognition [21, 47, 48]. Similarly, the predicted domains of EsSR-B1 also contain an

368

extracellular domain, two transmembrane regions and two cytoplasmic tails. Multiple

369

alignment of the amino acid sequence of EsSR-B1 showed relatively low

370

conservation of the SR-B family, but high homology with crustaceans from M.

371

japonicas. The phylogenetic tree revealed three distinct clades, including SR-As,

372

SR-Bs and SR-Cs. EsSR-B1 belongs to a subgroup of SR-Bs. Therefore, the

373

relationships in the phylogenetic tree corresponded to their taxonomic classifications.

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SR-Bs was found to be mainly distributed in immune organs and hematopoietic

375

cells such as platelets, monocytes and macrophages [22], while little is known about

376

SR-Bs in crustacean. In this study, the tissue distribution of EsSR-B1 was analyzed by

377

qRT-PCR, which revealed that it was expressed in hemocytes, heart, hepatopancreas, 18

ACCEPTED MANUSCRIPT gills, stomach and intestine. SR-Bs showed important roles in the host immune

379

response to fungi and bacteria and could bind erythrocytes infected with the malaria

380

parasite Plasmodium falciparum [49, 50]. In our study, similarly, EsSR-B1 was found

381

to be upregulated in hemocytes after challenge with Gram-positive and

382

Gram-negative bacteria, suggesting its potential role in innate immunity.

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Phagocytosis is an evolutionarily conserved process involved in endocytosing and

384

destroying cellular debris and pathogens [1, 10]. PRRs, opsonic receptors and

385

apoptotic corpse receptors are related to phagocytosis, which have been identified in

386

humans [51]. The process of phagocytosis begin with pathogens recognizing by PRRs

387

and adhesion of the pathogens to the phagocyte surface [7, 10]. Pathogen recognition

388

and internalization is mediated by various phagocytic receptors, including Fcγ

389

receptor, integrins (complement receptors), scavenger receptors, and C-type lectins

390

such as mannose binding receptor in mammals [52, 53]. In higher species, phagocytes

391

function as important antigen-presenting cells and are required to prime effective

392

adaptive immunity [54]. During pathogenic invasion, phagocytosis is also the

393

cornerstone of the early innate immune response and host defense mechanisms of

394

many species [55]. In Chinese mitten crab, C-type lectins (CTLs) has been identified

395

as one of PRRs. It strongly binds to Gram-negative bacteria, Gram-positive bacteria

396

and fungi [56]. There are also some other PRRs identified in Chinese mitten crab,

397

such as Toll-like receptors (TLRs) [57] and Dscam, which may be associated with

398

phagocytosis. SRs, kind of PRRs, are worth to be studied for their phagocytosis. In

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19

ACCEPTED MANUSCRIPT previous reports, many SR-Bs involved in phagocytosis were identified in other

400

invertebrates, such as M. japonicas [25] and D. melanogaster [58]. A SR-B (MjSR-B1)

401

was suggested to be able to bind to Gram-positive and Gram-negative bacteria in M.

402

japonicas. Further study revealed that MjSR-B1 could protect shrimp from bacteria by

403

promoting phagocytosis, and this ability was weakened by knockdown of MjSR-B1

404

[25]. In this study, the hemocytically expressed EsSR-B1 recognizes bacteria and

405

enhances phagocytosis of invading bacteria. Therefore, like mammals, Chinese mitten

406

crabs also have phagocytic receptors (e.g., EsSR-B1) that directly recognize the

407

pathogens. This also indicates that phagocytosis plays a vital role in innate immunity.

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In the current study, our results revealed that EsSR-B1 participates in the

409

regulation of the expression of AMPs in hemocytes. SR-Bs include receptors recently

410

known as SR-BI, SR-BII, CD36 and LIMP2 in mammals [22].In a previous study,

411

upon stimulation with diacylglycerides, CD36 was found to associate with TLR2/6

412

heterodimers in lipid rafts at the cellular surface, and this interaction was considered

413

to be vital for signaling in response to microbial molecules [59]. CD36 could also

414

cooperate with a TLR4/6 heterodimer, which occurred following sterile inflammation,

415

such as that mediated by oxidized low-density lipoprotein (OxLDL). The MyD88 and

416

TRIF adaptors were shown to transmit TLR4–TLR6 signaling, leading to the

417

induction of pro-inflammatory cytokines [60]. The induction of AMPs against

418

bacteria might be via TLR pathways. The relationship between EsSR-B1 and TLR

419

pathways needs to be further studied and explored.

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5. Conclusions Taken together, the results from this work focus the role of a pattern recognition

423

receptor EsSR-B1 in a crustacean Eriocheir sinensis. In this work, we obtained and

424

identified the full-length cDNA of the EsSR-B1 gene from E. sinensis. The

425

transcriptional levels showed that EsSR-B1 was highly expressed in intestine and

426

hepatopancreas, as well as hemocytes. In addition, EsSR-B1 was upregulated

427

significantly in hemocytes after bacterial stimulation. Moreover, the extracellular

428

region of EsSR-B1 was shown to possess the ability to bind and agglutinate bacteria.

429

We also found that EsSR-B1 could act as a phagocytic receptor for both

430

Gram-positive and Gram-negative bacteria to affect phagocytosis and serve as a PRR

431

to regulate their expression. Thus, EsSR-B1 has a critical role in the binding of

432

bacteria and subsequent promotion of hemocyte phagocytosis

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Acknowledgements

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This work was supported by grants from National Natural Science Foundation of

436

China (31602189, 31672639), Shanghai Natural Science Foundation (15ZR1410800)

437

and the Shanghai Collaborative Innovation Center for Aquatic Animal Genetics and

438

Breeding, the Opening Project of Key Laboratory of Freshwater Fishery Germplasm

439

Resources, Ministry of Agriculture, P. R. China, and the Shanghai University

440

Knowledge Service Platform, Shanghai Ocean University Aquatic Animal Breeding 21

ACCEPTED MANUSCRIPT 441

Center (ZF1206).

442

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Figure legends 625

Figure 1. Nucleotide and deduced amino acid sequences of EsSR-B1. The amino

626

acid sequences are shown under the cDNA sequences. The CD36 domain is

627

underlined. Transmembrane (TM) segments are shaded in dark gray, while letters

628

representing start codons (ATG), stop codons (TAG) and polyadenylation site

629

(AATAAA) are bolded. 30

ACCEPTED MANUSCRIPT Figure 2. A schematic view of the structure of the EsSR-B1 protein and its

631

three-dimensional model. (A) Domain analysis of the putative EsSR-B1 protein by

632

the SMART tool showing that it contains a CD36 domain. (B) Predicted

633

transmembrane regions of EsSR-B1 by the TMHMM Server. The two transmembrane

634

regions are located at the N- and C-terminals of the protein. (C) A three-dimensional

635

model of the EsSR-B1 built by the online software SWISS-MODEL. The template ID

636

is 4q4b1.

637

Figure 3. Multiple sequence alignment of EsSR-B1 with SR-B from other species.

638

The sequences and accession numbers of proteins used for alignment are as follows:

639

M. japonicas AKO62849, Bos mutus XP_005899218, Rhinolophus hipposideros

640

AFI71420,

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NP_001117983, Oplegnathus fasciatus BAM36398, B. japonicum AEY79768, O.

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cincta ODN04768. Numbers refer to residues counted from the initiation methionine.

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Residues identical in sequences are marked in black.

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Figure 4. NJ phylogenetic analysis of SR proteins. The NJ phylogenetic tree was

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constructed with the sequence analysis tool MEGA 6. The red triangle (▲) indicates

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EsSR-B1. The sequences and their accession numbers are as follows: Homo sapiens

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NP_001076428, Rattus norvegicus BAA14004, Chelonia mydas EMP26516,

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Nanorana parkeri XP_018413132, Nothobranchius furzeri SBP55920, Daphnia

649

magna JAN13991, O. cincta ODN04768, M. japonicas AKO62849, M. japonicas

650

BAJ10664, O. cincta ODN03822, Culex quinquefasciatus EDS37532, Tribolium

niloticus

XP_003444373,

Oncorhynchus

mykiss

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Oreochromis

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ACCEPTED MANUSCRIPT castaneum EFA02814, D. melanogaster NP_787957, Lygus hesperus JAQ16620,

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Pediculus humanus corporis XP_002427890, Anopheles darling ETN63673, D.

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melanogaster NP_001285599, D. magna JAN65020, Danio rerio NP_001025361,

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Charadrius vociferus KGL88332, Bos taurus NP_001095969, R. norvegicus

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NP_001129327, H. sapiens NP_776194, Macaca mulatta AFJ70870.

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Figure 5. Tissue-specific expression patterns of EsSR-B1. Tissue distribution

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analysis of EsSR-B1 expression in unchallenged crab by qRT-PCR. β-actin

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transcription was used as the control. The X-axis represents different tissues, and the

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Y-axis represents corresponding relative expression levels. The assay was performed

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three times, and data were analyzed by the unpaired t-test. *P < 0.05, **P < 0.01,

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compared with control.

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Figure 6. Expression pattern of EsSR-B1 in hemocytes. Expression patterns of

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EsSR-B1 in hemocytes after challenge by V. anguillarum or S. aureus as determined

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by qRT-PCR. The X-axis represents the time post challenge, and the Y-axis represents

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corresponding relative expression levels. All assays were performed three times, and

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data were analyzed by the unpaired t-test. *P < 0.05, **P < 0.01, compared with

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control.

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Figure 7. Expression and purification of extracellular region of EsSR-B1. The

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prokaryotic expression of EsSR-B1 was analyzed by 12% SDS-PAGE. Lane M,

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protein marker; lane 1, proteins of normal E. coli containing pET-28a-EsSR-B1; lane

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2, proteins of E. coli pET-28a-EsSR-B1 after induction by 0.25 mM IPTG; lane 3,

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ACCEPTED MANUSCRIPT purified rEsSR-B1; lane 4, rEsSR-B1 after renaturation.

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Figure 8. Binding of rEsSR-B1 to different bacteria. Bacteria were incubated with

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rEsSR-B1 and then washed with TBS three times. The precipitated bacteria samples

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were separated by SDS–PAGE and transferred to PVDF membranes for Western blot

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analysis. An anti-His-tag monoclonal antibody was used in the Western blot.

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Figure 9. EsSR-B1-mediated phagocytosis of bacteria by hemocytes in crab. (A)

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The effect of siRNA silencing on transcription of EsSR-B1 in hemocytes of E. sinensis.

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Twenty-four hours after the transfection of siEsSR-B1, hemocytes were collected for

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RNA extraction, and the EsSR-B1 mRNA expression level was analyzed by qRT-PCR.

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siGFP was used as the control. Data were analyzed by the unpaired t-test, and

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significant differences were accepted at P < 0.05 (B) Phagocytosis of heat-killed,

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FITC-labeled bacteria by hemocytes from E. sinensis. Twenty-four hours after

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siEsSR-B1 transfection, FITC-labeled S. aureus or V. parahaemolyticus (green) was

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added to hemocytes. The hemocytes were collected 1.5 h later and stained with DAPI

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(blue) and then observed under a fluorescence microscope. Hemocytes treated with

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siGFP were used as controls. Scale bar = 5 µm. (C) The phagocytosis rate was

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calculated using images captured by the fluorescence microscope, and a total of 1000

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cells were counted. Data were analyzed by the unpaired t-test, and significant

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differences were accepted at P < 0.05. (D) Flow cytometry was used for hemocyte

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phagocytotic analysis. After drawing the gate, the phagocytic rate was determined and

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displayed in the upper right corner of the image. The green background represents the

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ACCEPTED MANUSCRIPT hemocytes which endocytosed the FITC-labeled bacteria.

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Figure 10. Expression analysis of AMPs in EsSR-B1-silenced hemocytes

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challenged by bacteria. The hemocytes with EsSR-B1 knockdown were challenged

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by (A) S. aureus and (B) V. parahaemolyticus, and then the expression levels of seven

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AMP genes (ALF1, 2 and 3, Crus 1 and 2, DWD and Lys) were analyzed by qRT-PCR.

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All assays were performed three times, and data were analyzed by the unpaired t-test.

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Significant differences were accepted at P < 0.05.

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ACCEPTED MANUSCRIPT

Table 1 Primer sequences used for EsSR-B1 analysis. Primer name Sequence (5’-3’) cDNA cloning EsSR-B1-5’ RACE GCTTCGCACCCAGGCTCATC EsSR-B1-3’ RACE CCCGTGTTTTGGGTCAACGAGAGC Real-time quantitative PCR β-actin qF GCATCCACGAGACCACTTACA β-actin qR CTCCTGCTTGCTGATCCACATC SR-B1 qF CGTGAGGGAACTCCTATTTG SR-B1 qR CCGTGTAGACCGTGTAAGC DWD1 qF ACGGGTCGTCAACGAAACTG DWD1 qR GGTCACTGGGTTACCATAGCG Lys qF CTGGGATGATGTGGAGAAGTGC Lys qR TTATTCGGTGTGTTATGAGGGGT ALF1 qF GACGCAGGAGGATGCTAAC ALF1 qR TGATGGCAGATGAAGGACAC ALF2 qF GACCCTTTGCTGAATGCTTGA ALF2 qR CTGCTCTACAATGTCGCCTGA ALF3 qF ACGAGGAGCAAGGAAAGAAAG ALF3 qR TTGTGCCATAGACCAGAGACTT Crus-1 qF GCTCTATGGCGGAGGATGTCA Crus-1 qR CGGGCTTCAGACCCACTTTAC Crus-2 qF GCCCACCTCCCAAACCTAT Crus-2 qR GCAAGCGTCACAGCAGCACT Prokaryotic expression EsSR-B1 YF CCGGAATTCGATCGCCTCTTCGACTCAATG EsSR-B1 YR CCGCTCGAGGAAGGGCAGGTTAAGGGTG RNA interference siEsSRB1 F CCGGAAGAACAACACGAAUTT siEsSRB1 R AUUCGUGUUGUUCUUCCGGTT

703

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EP

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ACCEPTED MANUSCRIPT Research highlights: 1. EsSR-B1 has broad binding abilities with bacteria. 2. EsSR-B1 is a phagocytic receptor for both Gram-positive and Gram-negative

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bacteria.

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3. EsSR-B1 can regulate the expression of antimicrobial peptides.