TMT-based quantitative proteomic analysis of Eriocheir sinensis hemocytes and thoracic ganglion during Spiroplasma eriocheiris infection

Journal Pre-proof TMT-based quantitative proteomic analysis of Eriocheir sinensis hemocytes and thoracic ganglion during Spiroplasma eriocheiris infection Libo Hou, Haifeng Zhou, Hui Wan, Zhanghuai Liu, Li Wang, Yongxu Cheng, Xugan Wu, Wei Gu, Wen Wang, Qingguo Meng PII:

S1050-4648(19)31052-6

DOI:

https://doi.org/10.1016/j.fsi.2019.11.009

Reference:

YFSIM 6578

To appear in:

Fish and Shellfish Immunology

Received Date: 5 September 2019 Revised Date:

29 October 2019

Accepted Date: 2 November 2019

Please cite this article as: Hou L, Zhou H, Wan H, Liu Z, Wang L, Cheng Y, Wu X, Gu W, Wang W, Meng Q, TMT-based quantitative proteomic analysis of Eriocheir sinensis hemocytes and thoracic ganglion during Spiroplasma eriocheiris infection, Fish and Shellfish Immunology (2019), doi: https:// doi.org/10.1016/j.fsi.2019.11.009. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Published by Elsevier Ltd.

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TMT-based quantitative proteomic analysis of Eriocheir sinensis hemocytes and

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thoracic ganglion during Spiroplasma eriocheiris infection

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Libo Hou a, 1, Haifeng Zhou a, 1, Hui Wan a, Zhanghuai Liu a , Li Wang b, Yongxu Cheng c,

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Xugan Wu c, Wei Gu a, d, Wen Wang a, *, Qingguo Meng a, d, *,

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a

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College of Marine Science and Engineering, Nanjing Normal University, 1 Wenyuan Road,

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Nanjing 210023, China

Jiangsu Key Laboratory for Aquatic Crustacean Diseases, College of Life Sciences &

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b

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China

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c

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d

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Lianyungang, Jiangsu, China

College of Life Science and Technology, Southwest Minzu University, Chengdu 610041,

Shanghai Ocean University, Shanghai, China Co-Innovation Center for Marine Bio-Industry Technology of Jiangsu Province,

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These authors contributed equally to this paper.

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*Corresponding authors: Qingguo Meng, Wen Wang, Jiangsu Key Laboratory for Aquatic

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Crustacean Diseases, College of Life Sciences & College of Marine Science and Engineering,

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Nanjing Normal University, 1 Wenyuan Road, Nanjing 210023, PR. China.

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E-mail addresses: [email protected] (Qingguo Meng), [email protected] (Wen Wang)

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ABSTRACT

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Spiroplasma eriocheiris, a novel pathogen of Chinese mitten crab Eriocheir sinensis

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tremor disease, has led into catastrophic economic losses in aquaculture. S. eriocheiris

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invaded the hemocytes in the early stage, then invaded nerve tissue and caused typically

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paroxysmal tremors of pereiopod in the late stage of infection. The purpose of this study was

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to detect the infection mechanism of hemocytes in the early stage and thoracic ganglion in the

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late stage of S. eriocheiris infection at the protein level. Hemocytes and thoracic ganglion

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were collected at 24 h and 10 d after injection (the crabs with typical paroxysmal tremors of

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the pereiopod), respectively. TMT was performed with isobaric markers, followed by liquid

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chromatography tandem mass spectrometry (LC-MS /MS). In hemocytes, 127 proteins were

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up-regulated and 85 proteins were down-regulated in 2747 quantified proteins. Many proteins

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and process including proPO system proteins, hemolymph coagulation system proteins and

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lectins were differently expressed in hemocytes and involved in the early immune process of

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E. sinensis against S. eriocheiris infection. Meanwhile, 545 significantly different expression

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proteins (292 down-regulated and 253 up-regulated protein including a number of

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immune-associated, nervous system development and signal transmission related proteins)

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were identified in thoracic ganglion in the late stage of S. eriocheiris infection. The qRT-PCR

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analysis results shown that the selected significantly changed proteins in hemocytes and

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thoracic ganglion were consistent with the TMT proteomics. This paper reported for the first

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time to study the responses of crab hemocyte and thoracic ganglion against the S. eriocheiris

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infection at different stages. These findings help us understand the infection mechanism of S.

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eriocheiris at different stage with the different tissue.

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Keyword: Spiroplasma eriocheiris; Eriocheir sinensis; hemocytes; thoracic ganglion

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1. Introduction

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Chinese mitten crab, Eriocheir sinensis, is a unique Chinese aquaculture species with

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important economic value. With the large-scale development of crab aquaculture industry,

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various diseases have flourished and caused serious economic losses. Among the numerous

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diseases of E. sinensis, tremor disease (TD) is the most common and devastating disease.

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Previous studies have shown that Spiroplasma eriocheiris is pathogenic bacteria of the E.

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sinensis TD [1,2]. Spiroplasma is a minimal prokaryotic microorganism and intracellular

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bacteria that lacks cell walls [3]. The infection characteristic showed that the hemocytes of

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crab as the first target cell of S. eriocheiris can form inclusion bodies to restrict the

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transference of S. eriocheiris [4]. Due to reproducation of S. eriocheiris, the inclusion bodies

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enlarged and destroy the hemocytes. Though circulation of blood, S. eriocheiris transferred

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and infected the thoracic ganglion of crab at the later stage [2]. The thoracic ganglion could

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control the pereiopod movement, so the S. eriocheiris infected the thoracic ganglion of crab

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were believed the directly reasons of caused symptoms of TD. However, the pathogenesis of

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E. sinensis TD at the different stage of S. eriocheiris infection with the different tissue still

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don’t known.

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To study the immune function in the defense system of crustaceans, many approaches

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have been applied. As one of the most effective analysis methods, proteomics has many

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advantages because it could be used to describe more direct molecular responses than gene

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level analysis [5]. Therefore, invertebrate proteomic analysis has been increasingly studied

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during the last few years to shed new light on the immune responses of host against pathogens

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infection. The proteomic analysis showed that amino acid metabolism, the tricarboxylic acid

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cycle and glycolytic pathway involved in the response of Penaeus vannamei against yellow

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head virus (YHV) infection [6]. By proteomic analysis, the lysozymes, lections and

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transthyretin-like proteins were participated in the process of Caenorhabditis elegans against

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Bacillus thuringiensis [7]. Proteomic analysis results shown that pattern recognition

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receptor-mediated immune responses and coagulation system were participated in Portunus

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trituberculatus against the Hematodinium infection [8]. By proteomic analysis, many pathway

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or proteins like proPO system, a2M, BGBP, LGBP and antimicrobial action (tachylectin,

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lectins, vitellogenin, etc.) were identified participated in Macrobrachium rosenbergii

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hemocytes against the S. eriocheiris infection [9]. iTRAQ analysis shown that the many

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immune-related proteins were identified play an important role in the process of Procambarus

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clakii resistance S. eriocheiris, such as serine protease, peroxiredoxin 6, 14-3-3-like protein,

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C-type lectin, cdc42 homolog precursor etc [10]. All these studies indicated that proteomic

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analysis was more accurate and reliable methods to help us better understand interaction

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between the invertebrates and pathogens.

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In this study, the proteomic analysis of E. sinensis hemocyte and thoracic ganglion to

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elucidate the infection characteristics of S. eriocheiris at the different infection stages and the

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pathogenesis of crab TD. The proteome of E. sinensis thoracic ganglion is the first proteome

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of invertebrate tissue. As the first target cell of S. eriocheiris, hemocyte differently expressed

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many immune proteins or relevant proteins to against this pathogen infection. The thoracic

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ganglion played an important role in control the pereiopod movement, so many immune and

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nervous system development or signal transmission related proteins would disorder when the

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S. eriocheiris infected to cause typically paroxysmal tremors of pereiopod. These results

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provide important new information about spiroplasma infection and the reason of crab

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pereiopod paroxysmal tremors.

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2. Materials and methods

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2.1. Experimental bacterial infection and tissue sampling

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S. eriocheiris was isolated from the diseased E. sinensis using the methods described by

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Wang et al., [11] and cultured in R2 medium at 30

. E. sinensis (50±3 g) were purchased

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from an aquaculture pond in Baoying, Jiangsu Province, China and cultivated in an ultraviolet

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radiation sterilization circulating water temperature controlled aquaculture system. Healthy E.

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sinensis (verified by S. eriocheiris negative results using PCR by 16s rDNA sequence

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analyses and hemolymph TEM negative staining methods) were maintained for 1 week before

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tests [12].

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The crabs in the experimental group (50 individuals) received an injection of 100 µL S.

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eriocheiris (107 cells/ml), individually. Fifty crabs in the control group received an injection

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of 100 µL R2 medium. Ten crabs from the control group and ten crabs from the experimental

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group at 24 h post-injection were randomly collected to prepare hemocyte samples. From

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previous studies, the S. eriocheiris begin to reproduce greatly in hemocyte from 24 h after

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injection [13]. The hemocyte was drawn from crabs using a 1-mL syringe, and quickly added

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into anticoagulant solution at a ratio of 1:1. Sterile anticoagulant citrate dextrose solution B

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(ACD-B, glucose, 1.47 g; citrate, 0.48 g; sodium citrate, 1.32 g; pH = 4.0; prepared in double

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distilled water at 100 mL final volume, and filtered with 0.22 µM filter) was employed.

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Samples were immediately centrifuged at 4000 rpm, 4

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Ten crabs in the control group and 10 crabs in the experimental group (with typical

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paroxysmal tremors of the pereiopod) were collected about 10 d after injection to prepare

for 5 min to collect the hemocytes.

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thoracic ganglion samples. Thoracic ganglion tissue is removed using a treated anatomical

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tool（Sterile and DEPC H2O treated scissors and tweezers） and immediately put into liquid

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nitrogen.

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2.2. Protein preparation

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After grinded by liquid nitrogen, the samples was transferred to 5 mL centrifuge tube

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and sonicated three times on ice using a high intensity ultrasonic processor (Scientz) in lysis

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buffer (8 M urea, 2 mM Ethylene Diamine Tetraacetic Acid, EDTA, 10 mM

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DL-Dithiothreitol, DTT and 1% Protease Inhibitor Cocktail). The remaining debris was

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removed by centrifugation at 20,000g at 4 °C for 10 min. Finally, the protein was precipitated

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with cold 15% Triethylammonium bicarbonate buffer, TCA for 2 h at -20 °C. After

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centrifugation at 4 °C for 10 min, the supernatant was discarded. The remaining precipitate

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was washed with cold acetone for three times. The protein was redissolved in buffer (8 M

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urea, 100 mM tetraethyl-ammonium bromide, TEAB, pH 8.0) and the protein concentration

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was determined with 2-D Quant kit (Biyuntian Company, China) according to the

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manufacturer’s instructions. For digestion, trifluoroacetic acid the protein solution was

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reduced with 10 mM DTT for 1 h at 37 °C and alkylated with 20 mM iodoacetamide (IAA)

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for 45 min at room temperature in darkness. For trypsin digestion, the protein sample was

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diluted by adding 100 mM TEAB to urea concentration less than 2 M. Finally, trypsin was

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added at 1:50 trypsin-to-protein mass ratio for the first digestion overnight and 1:100

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trypsin-to-protein mass ratio for a second 4 h-digestion. Approximately 100 µg protein for

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each sample was digested with trypsin for the following experiments.

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2.3. TMT labeling and HPLC fractionation

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After trypsin digestion, peptide was desalted by Strata X C18 SPE column (Phenomenex)

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and vacuum-dried. Peptide was reconstituted in 0.5 M TEAB and processed according to the

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manufacturer’s protocol for 6-plex TMT kit. Briefly, one unit of TMT reagent (defined as the

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amount of reagent required to label 100 µg of protein) were thawed and reconstituted in 24 µl

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acetonitrile (ACN). The peptide mixtures were then incubated for 2 h at room temperature

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and pooled, desalted and dried by vacuum centrifugation. The samples were then fractionated

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into fractions by high pH reverse-phase HPLC using Agilent 300Extend C18 column (5 µm

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particles, 4.6 mm ID, 250 mm length). Briefly, peptides were first separated with a gradient of

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2% to 60% acetonitrile in 10 mM ammonium bicarbonate pH 10 over 80 min into 80 fractions.

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Then, the peptides were combined into 18 fractions and dried by vacuum centrifuging.

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2.4. LC-ESI-MS/MS analysis

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Peptides were dissolved in 0.1% FA, directly loaded onto a reversed-phase pre-column

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(Acclaim PepMap 100, Thermo Scientific). Peptide separation was performed using a

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reversed-phase analytical column (Acclaim PepMap RSLC, Thermo Scientific). The gradient

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was comprised of an increase from 6% to 10% solvent B (0.1% FA in 98% ACN) over 4 min,

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10% to 23% in 22 min, 23% to 36% in 8 min and climbing to 85% in 5 min then holding at

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85% for the last 3 min, all at a constant flow rate of 300 nl/min on an EASY-nLC 1000 Ultra

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Performance Liquid Chromatography (UPLC) system. The peptides were analyzed by Q

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ExactiveTM plus hybrid quadrupole-Orbitrap mass spectrometer (ThermoFisher Scientific).

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The peptides were subjected to nano electrospray ionization (NSI) source followed by

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tandem mass spectrometry (MS/MS) in Q ExactiveTM plus (Thermo) coupled online to the

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UPLC. Intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were

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selected for MS/MS using normalize crash energy (NCE) setting as 27, 30, 33; ion fragments

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were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure that

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alternated between one MS scan followed by 20 MS/MS scans was applied for the top 20

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precursor ions above a threshold ion count of 2E4 in the MS survey scan with 30.0 s dynamic

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exclusion. The electrospray voltage applied was 2.0 kV. Automatic gain control (AGC) was

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used to prevent overfilling of the orbitrap; 5E4 ions were accumulated for generation of

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MS/MS spectra. For MS scans, the m/z scan range was 350 to 1800. Fixed first mass was set

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as 100 m/z.

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2.5. Database Search

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The resulting MS/MS data were processed using Mascot search engine (v.2.3.0).

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Tandem mass spectra were searched against transcriptome database (paper under review).

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Trypsin/P was specified as cleavage enzyme allowing up to 2 missing cleavages. Mass error

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was set to 10 ppm for precursor ions and 0.02 Da for fragment ions. Carbamidomethyl on Cys,

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were specified as fixed modification and oxidation on Met was specified as variable

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modifications. For protein quantification method, TMT-6-plex was selected in Mascot. FDR

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was adjusted to < 1% and peptide ion score was set ≥ 20.

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2.6 Bioinformatics Analysis

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Enrichment of Gene Ontology analysis

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Proteins were classified by GO annotation into three categories: biological process,

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cellular compartment and molecular function. For each category, a two-tailed Fisher’s exact

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test was employed to test the enrichment of the differentially expressed protein against all

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identified proteins. Correction for multiple hypothesis testing was carried out using standard

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false discovery rate control methods. The GO with a corrected p-value < 0.05 is considered

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significant. The version for database used in this paper is InterProScan/GO v.5.14-53.0

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(http://www.ebi.ac.uk/interpro/), the cutoff for sequences similarity is E-value < 1e-10.

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Enrichment of pathway analysis

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Encyclopedia of Genes and Genomes (KEGG) database was used to identify enriched

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pathways by a two-tailed Fisher’s exact test to test the enrichment of the differentially

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expressed protein against all identified proteins. Correction for multiple hypothesis testing

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was carried out using standard false discovery rate control methods. The pathway with a

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corrected p-value < 0.05 was considered significant. These pathways were classified into

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hierarchical categories according to the KEGG website. The version for database used in this

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paper is KEGG v.2.0 (http://www.genome.jp/kaas-bin/kaas_main).

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Enrichment of protein domain analysis

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Protein domain analysis were annotated by InterProScan (http://www.ebi.ac.uk/interpro/,

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v.5.14-53.0) (a sequence analysis application) based on protein sequence alignment method.

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For each category proteins, InterProScan (a resource that provides functional analysis of

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protein sequences by classifying them into families and predicting the presence of domains

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and important sites) database was researched and a two-tailed Fisher’s exact test was

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employed to test the enrichment of the differentially expressed protein against all identified

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proteins. Correction for multiple hypothesis testing was carried out using standard false

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discovery rate control methods and domains with a corrected p-value < 0.05 were considered

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significant.

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2.7. Real-time PCR

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The samples of crab hemocytes and thoracic ganglion used to Real-time PCR (qRT-PCR)

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were same with the proteomic analysis. After extraction, total RNA was reverse-transcribed

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into cDNA with a PrimeScript RT reagent Kit (TAKARA, Japan). The partial sequences of

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genes were amplified by primers listed in Table 1. GAPDH was used as a housekeeping gene.

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The qRT-PCR reaction was performed in a 20 µL volume with a SYBR Premix Ex Taq™ Kit

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(Takara, Japan), 2 µM of each specific primer and 1 µL of cDNA in Mastercycler ep realplex

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using the following procedure: initial denaturation at 95

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amplification (95

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of different genes in hemocytes and thoracic ganglion were calculated according to the 2−∆∆CT

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method. Statistical analysis was performed using SPSS software (Ver11.0). Data represent the

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mean ± standard error (S.E.). Statistical significance was determined by one-way ANOVA,

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and Duncan multiple range tests. Significance was set at p < 0.05.

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3. Results

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3.1. Protein profiling

for 10 s, 55

for 30 s, and 72

for 2 min, followed by 40 cycles of

for 30 s). The relative expression levels

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All MS/MS spectra were processed using Mascot software. A total of 3,192 proteins

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were identified in this hemocytes sample, of which 2,747 contained quantitative information.

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For thoracic ganglion, a total of 3,650 proteins were identified, of which 2,387 contained

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quantitative information. GO, KEGG, protein domain analysis and subcellular localization

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were used to analysis of total proteins in hemocytes and thoracic ganglion (Table S1 and

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Table S2). Using a 1.2-fold decrease or increase in proteins expression as a benchmark for

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physiologically significant change. In hemocytes, 212 differentially expressed proteins were

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reliably quantified by TMT analysis (Table S3), including 127 up-regulated proteins and 85

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down-regulated proteins subsequent to S. eriocheiris infection. Of the up-regulated proteins,

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there were many proteins involved in immune response of the host cell, including hemocytin,

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glutathione S-transferase (GST), clotting factor B/B2, melanization protease 1, serine protease

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inhibitor, cathepsin L, etc. In these down-regulated proteins, there also many proteins were

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grouped within the immune system proteins including lectin, lipopolysaccharide and

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beta-1,3-glucan-binding protein (LGBP), ferritin2/3, lysozyme C. The representative

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significantly different expression proteins identified in E. sinensis hemocytes proteome and

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the corresponding TMT ratios are presented in Table 2.

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For thoracic ganglion, in total 545 differentially expressed proteins were reliably

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quantified by TMT analysis (Table S4), including 253 up-regulated proteins and 293

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down-regulated proteins subsequent to S. eriocheiris infection. In the up-regulated expression

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proteins including many immunity proteins and nervous system associated proteins, such as

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alpha-2-macroglobulin 2, hemocyanin subunit 6, heat shock proteins, neuronal calcium sensor

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2, synaptotagmin 2, etc. In the down-regulated proteins, also have many immunity proteins

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and nervous system associated proteins were identified, such as Rab GTPases family prteins,

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β-catenin, laccase-4, synaptosome-associated protein of 25 kDa (SNAP-25), calcium channel

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flower (CCF). The representative significantly expression proteins identified in E. sinensis

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thoracic ganglion proteome and the corresponding TMT ratios are presented in Table 3.

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3.2. GO enrichment

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GO annotation enrichment was used to analysis the differentially expressed proteins in

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hemocytes and thoracic ganglion (Fig 1, Table S5 and Table S6). For hemocytes (Fig. 1A,

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Table S5), many biosynthetic process were enrichment from biological process ontology

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perspective. For molecular function perspective, structural molecule activity, iron ion binding,

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oxidoreductase activity, etc. were enrichment in hemocytes. For cellular component,

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non-membrane-bounded

organelle,

intracellular

non-membrane-bounded

organelle,

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protein-DNA complex, etc. were enrichment in hemocytes. For thoracic ganglion (Fig. 1B，

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Table S6), chitin metabolic process, microtubule-based process, GDP-mannose biosynthetic

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process, etc. were significantly enrichment base on biological process analysis. From the

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molecular function perspective, calcium ion binding, chitin binding, GTPases activity, etc.

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were enrichment in thoracic ganglion. For cellular component, extracellular region,

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proteinaceous extracellular matrix and extracellular region part were significantly enrichment

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in thoracic ganglion.

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3.3. Subcellular Localization

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Subcellular localization analysis was used to annotate the differently expression proteins

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identified in this paper. For hemocytes (Fig. 2A), the proportion of each component is

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cytoplasm (32%), extracellular (23%), nucleus (19%), plasma membrane (11%),

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mitochondria (6%). For thoracic ganglion (Fig. 2B), the proportion of each component is

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Cytosol (28%), nuclear (19%), extracellular (18%), plasma membrane (13%).

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3.4. KEGG Enrichment

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KEGG enrichment were used to analysis the differentially expressed proteins in

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hemocytes and thoracic ganglion of crab related to the S. eriocheiris infection. For the

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hemocytes (Fig. 3A, Table S7), ribosome, steroid biosynthesis and aminoacyl-tRNA

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biosynthesis were significantly enrichment in the up-regulated proteins. In the down-regulated

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proteins, drug metabolism - cytochrome P450, metabolism of xenobiotics by cytochrome

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P450, glutathione metabolism, etc. were significantly enrichment in the hemocytes. For

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thoracic ganglion (Fig. 3B, Table S8), one carbon pool by folate, ECM-receptor interaction

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and lysine degradation were significantly enrichment in the up-regulated proteins. For the

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down-regulated proteins in thoracic ganglion, Wnt signaling pathway, mitophagy, endocytosis,

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phosphatidylinositol signaling system, etc. were enrichment.

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3.5. Protein domain enrichment

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To reveal the domain’s change of differently expreesion proteins, protein domain

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enrichment analysis of were carried out. In hemocytes (Fig. 4A, Table S9), nucleic

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acid-binding, cold-shock protein, Kazal domain, C-type lectin-like/link domain, C-type lectin

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fold, etc were significantly enriched in the up-regulated domains enrichment analysis. For the

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down-regulated domain enrichment, there also many immune-related domains were

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significantly enriched, such as C-lection domain (C-type lectin-like, C-type lectin-like/link

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domain, C-type lectin fold), ferritin-related domains (fferritin-related, ferritin-like superfamily,

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ferritin-like diiron domain and ferritin/DPS protein domain), immunoglobulin-like fold.

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For thoracic ganglion (Fig. 4B, Table S10), there were many domains were significantly

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up-regulated compare with the control group, including hemocyanin/hexamerin middle

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domain, immunoglobulin E-set, zinc finger LIM-type, hemocyanin, C-terminal, trypsin

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inhibitor-like. In the down-regulated domains, there also have many domains were

288

enrichment, such as many immune-related domains (immunoglobulin subtype, growth factor

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receptor cysteine-rich domain, immunoglobulin-like domain, immunoglobulin subtype 2,

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CD80-like, immunoglobulin C2-set, etc.), nervous system-related domains (SAC domain,

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nicotinic acetylcholine-gated receptor transmembrane domain, kinesin motor domain,

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EF-hand domain, neurotransmitter-gated ion-channel transmembrane domain, etc.).

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3.6. qRT-PCR analysis

294

In order to provide additional mRNA transcript levels of E. sinensis and validate the

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proteomics analysis result, qRT-PCR in both the hemocytes and thoracic ganglion were

296

performed. For the hemocytes, the mRNA transcription levels of nine proteins, including four

297

up-regulated proteins (cathepsin L, hemocytin, peroxiredoxin 1 (Prx1) and NADPH oxidase 5

298

(NOX5)) and four down-regulated proteins (LGBP, ferritin 2 (Fer2), Kazal-type protease

299

inhibitor (KPI) and VEGFR1) were measured. The qRT-PCR results shown that the

300

transcription tendencies of those selected genes in transcriptional level were consistent with

301

the TMT proteomics analysis (Fig. 5A).

302

For the thoracic ganglion, the mRNA transcription levels of twelve proteins, including

303

four up-regualted proteins (scavenger receptor (SR), prophenoloxidase activating enzyme III

304

(ppAIII), prophenoloxidase-activating factor (PPAF) and α-2-macrophageis (α2M)) and four

305

down-regulated proteins (CCF, SNAP25, Rab10 and CD63) were measured. As the results

306

shown in the Fig.5B, all the results of qRT-PCR analysis were consistent with the proteomics

307

analysis.

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4. Discussion

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In recent years, the occurrence of TD caused by S. eriocheirs in E. sinensis has caused

310

serious impacts on the aquaculture industry. The S. eriocheirs mainly infected hemocyte of

311

crab at the early stage, infected and damage the nervous system at the later stage. However,

312

the relationship between S. eriocheirs and crab in these two stages has not been studied. In

313

this article, the proteome of hemocytes at the early stages of S. eriocheirs infection (24 h) and

314

the proteome of thoracic ganglion after 10 d of S. eriocheirs infection (the crabs with typical

315

paroxysmal tremors of the pereiopod) were analyzed.

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4.1. Immune response of hemocytes at the early stages of S. eriocheirs infection

317

In the early stage of the S. eriocheiris infection, the hemocytes of the crab, as the main

318

target cell, was selected to study the relationship between S. eriocheiris and the host at the

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early stages using proteomics analysis. Many biological processes, pathways and domain

320

were enrichment participated in the process of S. eriocheiris infection, including steroid

321

biosynthesis, glutathione metabolism, C-type lectin domain, ferritin-related domain,

322

glutathione S-transferase domain, etc. These predicted biological processes, pathways and

323

domains can provide useful information for further investigation of gene functions.

324

Oxidoreductase system play an important role in host defence mechanism against microbial

325

infection. Glutathione S-transferase (GSTs), thioredoxin reductase (TrxR), Prx1 and NOX5

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belonged to the oxidoreductase system and involved in many cellular processes such as cell

327

growth and protection against oxidation stress. NADPH, TrxR and thioredoxin constitute the

328

Trx system is a key antioxidant system against oxidative stress. TrxR and NADPH oxidase

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activity were increased in Vibrio parahaemolyticus stimulated mud crabs, which was

330

consistent with the results of this study [14]. At the same time, the transcription of Prx1 and

331

NOX5 were also significantly up-regulated at the early stage of the S. eriocheiris infection.

332

GSTs, an essential part of cellular detoxification system, has the capability to protect the

333

organisms from the toxicity of reactive oxygen species (ROSs) caused by pathogen infection

334

or other stress. Similar to the early stage of S. eriocheiris infection in hemocyte of E. sinensis,

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the expression level of GST was down-regulated in M. rosenbergii under the stimulation of

336

Vibrio anguillaris [15].

337

The hemolymph coagulation of the humoral immune response is one of the most

338

important part of crustacean innate immune and plays an important role in the process of

339

defense against pathogens [16,17]. Coagulation-related proteins play a crucial role in this

340

process. In this study, the expression of clotting factor B1, clotting factor B2 and hemolymph

341

clottable protein in the hemocyte were significantly changed after S. eriocheiris infection,

342

indicating that the hemolymph coagulation also involved in the host immune response to S.

343

eriocheiris. Meanwhile, hemocytin molecule is an adhesive protein and relates to hemostasis

344

or encapsulation of foreign substances for self-defense [18]. As a non-specific immune factor,

345

hemocytin plays the similar role just like the complement system of vertebrates and activates

346

the lysis process to dissolve invading pathogen. Due to the infection of the S. eriocheiris, the

347

expression of hemocytin was increased in translational and transcription level. So hemocytin

348

was involved in the process of scavenging the bacteria.

349

The innate immune system is composed of pattern recognition receptors (PRRs), which

350

can recognize pathogen-associated molecular patterns (PAMPs) and activate the host immune

351

response. In this paper, several kinds of PRRs were identified, including mannose-binding

352

protein (MBP), LGBPand integrins etc. MBP is a PRR, which is one kinds of C-type lectin

353

and plays a crucial role in the innate immune response by recognizing and binding with the

354

PAMPs of the microbes and activity the host cell immune system, such as

355

prophenoloxidase-activating system (proPO system)[19]. Consist with the results in this paper,

356

the transcription of Portunus trituberculatus MBP also up-regulated against V. alginolyticus,

357

Micrococcus luteus or Pichia pastoris infection [20]. The proPO system, only found in the

358

invertebrates, is a most important part of invertebrate innate immune. When the pathogen

359

invading the host cell, the receptors would recognize the pathogen and then activate the

360

proPO system to eliminate the invading pathogen by melanization [21-23]. In this paper,

361

when the S. eriocheiris infected the hemocytes of crab, the expression of serpins

362

(melanization protease 1, serine proteinase and serpin 3) and serine protease inhibitors (serine

363

protease inhibitor and Kazal-type protease inhibitor) were significantly changed. This results

364

showed that the proPO system was also an important immune response of the host against S.

365

eriocheiris infection at the early stage, and the MBP may as the receptor to recognize this

366

bacterium. LGBP could recognize and bind the pathogen surface common epitopes such as

367

beta-1,3-glucans [24]. Our previous study about the process of S. eriocheiris infect the

368

hemocytes of M. rosenbergii shown that the LGBP was the receptor of this bacteria and help

369

this pathogen infection [25]. And the expression of LGBP in the M. rosenbergii hemocytes

370

was down-regulated after the S. eriocheiris infection [9]. In this study, the expression of

371

LGBP in transcription and translational level were down-regulated at the early stage of S.

372

eriocheiris infection. This was due to the fact that the crab’s LGBP was also the receptor of S.

373

eriocheiris. And the host reduced the invading bacteria though down-regulated the expression

374

of LGBP. Integrin is a kind of heterodimeric transmembrane protein consists of α and β

375

subtypes and adhesion receptors for extracellular ligands [26]. The pathogens can ligand

376

binding to host integrins and trigger an ‘outside-in’ signaling pathway in turn recruit a huge

377

network of proteins, at last lead to the local reorganization of the actin cytoskeleton help

378

themselves enter into host cell [27-30]. At the early stage of the S. eriocheiris infection, the

379

expression of integrin alpha-PS3 was decreased. Meanwhile, many cytoskeletal

380

reorganization related proteins had significantly changed, such as inverted formin-2, ras-like

381

GTP-binding protein (Rho) and actin. This results shown that when infected the host cell, the

382

S. eriocheiris would interact with integrins and trigger the reorganization of cytoskeleton to

383

help itself enter into the host cell.

384

The iron and the iron-related proteins were proved to be involved in the process of hosts

385

against pathogens invading. When the E. sinensis was supplied exogenous iron, the copy

386

number of S. eriocheiris in the hemocytes were also significantly reduced [31]. In this paper,

387

at the early stage of S. eriocheiris infection the expression of two ferritin proteins (ferritin 2

388

and ferritin 3) in hemocytes were significantly changed. This results further confirm that the

389

ferritin proteins were involved in the process of host cell against the pathogen invading.

390

Meanwhile, by domain enrichment analysis also found many ferritin-related domains were

391

have significantly changed, including ferritin-related domain, ferritin-like superfamily domain,

392

ferritin-like diiron domain etc.

393

4.2. Immune response of the thoracic ganglion at the late stages of S. eriocheirs infection

394

In addition to the hemocyte samples collected in the early stage of S. eriocheiris

395

infection, the thoracic ganglion of the crabs infected with S. eriocheiris at the late stage was

396

also collected for analysis. Many biological processes, pathways and domains were identified,

397

including EMC-receptor interaction, Wnt signaling pathway, Endocytosis, EGF-like domain,

398

EF-hand domain, etc. As one of the sub families of PRRs, SRs can recognize a wide types of

399

exogenous ligands, such as including modified lipoproteins, danger-associated molecular

400

patterns (DAMPs) [32]. The transcript levels of SRs were significantly up-regulated in mud

401

crabs hemocytes and hepatopancreas following challenge with V. parahaemolyticus, LPS,

402

WSSV or PolyI:C to promote host cell bacteria clearance by enhancing phagocytosis [33].

403

This is consistent with the expression of SRs in E. sinensis infected with S. eriocheiris no

404

matter in transcript level or translational level. E. sinensis SRs may recognize S. eriocheiris

405

and enhance phagocytosis of the host to kill the invaded pathogen. Lysosomes, as an acidic

406

organelle, play a crucial role in the degradation of phagocytic vacuoles, autophagic substrates

407

and macromolecules [34,35]. In this study, after the S. eriocheiris infection, three kinds of

408

acid hydrolase (lysosomal alpha-mannosidase-like, cathepsin C and cathepsin D) were

409

significantly up-regulated. Consist with this results, the crab cathepsin D had been proved to

410

play an important role in the process of host cell combat with invading S. eriocheiris [36].

411

This results suggested the host cell eliminated the invaded pathogen through up-regulated the

412

acid hydrolase expression when S. eriocheiris entered into the host cell. Meanwhile, Rab

413

GTPases proteins, as molecular switches, are important regulators of membrane traffic on the

414

biosynthetic and endocytic pathways [33,37]. In recent years, many Rab proteins had been

415

found to be involved in crustacean innate immunity. When the E. sinensis infected with

416

Vibrio anguillarum, the expression of EsRab3 in hemocytes was significantly up-regulated

417

[38]. The Penaeus japonicas Rab protein (PjRab) could regulate shrimp hemocytic

418

phagocytosis to against the WSSV infection [39, 40]. In this study, at the late stage of S.

419

eriocheiris infection, many Rabs or Rab-related proteins (Rab6-interacting family member 1,

420

Rab10, Rab6A, Rab-3 and Rab27A-GAP-beta) were significantly changed in the crab

421

thoracic ganglion. This results shown that the Rab GTPases family proteins may also played

422

essential roles in the immune response to S. eriocheiris infection in crab by phagocytosis.

423

Wnt signaling pathway plays important role in regulated the nerve system development

424

and the innate immune. β-Catenin is a key regulator of the Wnt signaling cascade. The Wnt

425

proteins interact with the receptors Frizzled (Fz) and initiate this signaling to inhibit the

426

degradation of β-catenin [41]. Accumulated β-catenin in the nucleus binds to the

427

transcriptional factor T cell factor/lymphoid enhancer factor (TCF/LEF), regulating the

428

transcription of multiple genes involved in cellular proliferation, differentiation, survival, and

429

apoptosis [42]. In Litopenaeus vannamei stimulated by WSSV, the expression of β-catenin

430

was down-regulated, which was consistent with the results in this paper [43]. Similarly,

431

coiled-coil domain-containing protein 6 (CCDC6), as a positive regulator of Wnt signaling,

432

was also down-regulated after the S. eriocheiris infection in thoracic ganglion. This results

433

shown that when the S. eriocheiris infect the thoracic ganglion, the Wnt signaling pathway

434

was restrained. At the same time, there were also many studies shown the Wnt signaling

435

pathway played a crucial role in neuronal differentiation, dendrite development, axon

436

outgrowth. The disorder of this pathway would induced several neurodegenerative diseases

437

[44, 45].

438

Similar with the results the S. eriocheiris infection hemocytes in this paper, the proPO

439

system also played an important role in thoracic ganglion against the S. eriocheiris infection.

440

At the later stage of the S. eriocheiris infection, many proPO system related proteins in

441

thoracic ganglion have significantly up-regulated, including PPAF, ppAIII, KPI and

442

pacifastin-like serine protease inhibitor. At the same time, the qRT-PCR results shown that

443

the mRNA transcript levels of PPAF and ppAIII were also up-regulated. At the same time,

444

there were also have many other immune-related proteins were identified. In invertebrates,

445

α2M, a broad range proteinase inhibitor, is crucially involved in many immune responses [46].

446

Similar to our result, the expression of α2M in Scylla serrata were up-regulated after WSSV

447

infection [47]. Heat shock proteins (HSPs) commonly exist in cells from both eukaryotic and

448

prokaryotic organisms with highly conserved in evolution. HSPs appear to play an important

449

role in responses to different stress, such as bacterial challenge in invertebrates [48,49]. At the

450

later stage of the S. eriocheiris infection, three kinds of HSPs (heat shock protein 21, heat

451

shock protein cognate 3 and heat shock protein 90) were significantly up-regulated in the

452

thoracic ganglion of the crab. This results shown the HSPs involved in the immune response

453

of host cell against S. eriocheiris infection.

454

4.3. Neural transduction of the thoracic ganglion at the late stages of S. eriocheirs infection

455

In addition to immune-related proteins, there were also significant changes in some

456

neuro-related proteins in the thoracic ganglion of E. sinensis infected with S. eriocheiris for

457

10 d, for example, synaptotagmin (Syt), semaphorin, calcium/calmodulin-dependent protein

458

kinase, astrocyte-derived neurotrophic factor, nicotinic acetylcholine receptor, SNAP25 and

459

vesicle-associated membrane protein (VAMP). Syt, the major calcium sensor for synaptic

460

vesicle exocytosis, participates in triggering neurotransmitter release at the synapse

461

[50].VAMP, a family of SNARE proteins, play an important role in the process of

462

neurotransmitter releasing [51,52]. Syt, SNAP25 and VAMP are the core proteins of

463

v-SNARE/t-SNARE complex, which plays critical role in trigger and adjustment of the target

464

membrane vesicles and fusion process to participate in the process of neurotransmitters and

465

hormones released strict control [53]. After S. eriocheiris infection, Syt, SNAP25 and VAMP

466

expression in thoracic ganglion were significantly decreased, which interfered with the

467

release of neurotransmitters. And the mRNA transcription level of SNAP25 was also

468

significantly down-regulated. Semaphorins are a family of secreted and transmembrane

469

proteins that play roles in axon guidance, organogenesis and cancer. Sema1A belongs to the

470

Sema family of axon guidance molecules that are well known for their roles in the regulation

471

of nervous system development in a variety of organisms [54]. In this study, the expression of

472

Sema1A in the thoracic ganglion was down-regulated after 10 d of S. eriocheiris infection,

473

indicated that the neurodevelopment of infected crabs was affected. Ca2+, as a second

474

messenger, contributes to the physiology and biochemistry of organisms and cell, such as

475

neurotransmitter release, contraction of muscle cell, regulated many enzymes activity [55,56].

476

The initiates release of neurotransmitters need Ca2+ entry through presynaptic voltage-gated

477

Ca2+ channels, so the Ca2+ regulation plays crucial roles in the neurotransmitters release. In

478

the current study, when the S. eriocheiris infected the thoracic ganglion, many Ca2+

479

regulate-related proteins were significantly changed, such as EF hand Ca-binding protein,

480

neuronal calcium sensor 2, neurocalcin homolog, CCF, calcium/calmodulin-dependent

481

protein kinase II (CaMKII), plasma membrane calcium ATPase, calcium-dependent secretion

482

activator. This results shown that the S. eriocheiris regulated those proteins expression to

483

disturb the Ca2+ regulation of the cell. The disturb of neurotransmitter metabolism could cause

484

many diseases. In our study, duo to the S. eriocheiris infected, many neurotransmitters related

485

proteins or receptors have were significantly changed in thoracic ganglion, including

486

acetylcholinesterase 1, nicotinic acetylcholine receptor, glutamate receptor, glutamine

487

synthetase 2. At the later stage of the S. eriocheiris infection, the infection caused many

488

nervous system development and signal transmission related proteins expression significantly

489

changed in the thoracic ganglion. And this may be the reasons of pereiopod paroxysmal

490

tremors in crab TD.

491

In conclusion, 121 differentially expressed proteins in the E. sinensis hemocyte after 24

492

h of S. eriocheiris infection and 546 differentially expressed proteins in the thoracic ganglion

493

of E. sinensis after 10 d of S. eriocheiris infection were found by proteomic analysis. From

494

these differentially expressed proteins, many differently expression immune-related proteins

495

and pathway were identified in hemocyte at the early stage of S. eriocheiris infection, such as

496

proPO system, clotting system, hemolytic, thioredoxin reductase, lectin, GST-D7 and LGBP.

497

Those proteins or pathways participated in the early immune response of host against S.

498

eriocheiris infection. On the other hand, when the S. eriocheiris infected the crab with typical

499

symptoms of appendage tremor, the proteins or related pathway including α2M, HSPs, Rabs,

500

SRs and Wnt signaling pathway were significantly changed and involved in the immune

501

process of crab thoracic ganglion against S. eriocheiris infection. At the same time, the

502

infection of S. eriocheiris has also changed the expression of CaMKII, CCF, SNAP25,

503

VAMP, Syt, nicotinic acetylcholine receptor and glutamate receptor. Then S. eriocheiris

504

infection changed the release of neurotransmitters, disturbed the never system and caused

505

pereiopod paroxysmal tremors of crab TD. All the results in the current study would help us

506

understand the processes of S. eriocheiris infection at different stage with the different tissue

507

and the pathogenesis of crab TD.

508

Acknowledgments

509

The current study was supported by the National Key Research and Development Program of

510

China (Grant No. 2018YFD0900602), the National Natural Science Foundation of China

511

(NSFC Nos. 31870168), the Modern Fisheries Industry Technology System Project of Jiangsu

512

Province (Grant No.JFRS-01) and the project funded by the Priority Academic Program

513

Development of Jiangsu Higher Education Institutions (PAPD).

514

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hsp70 in white shrimp, Litopenaeus vannamei in response to bacterial challenge. Journal of

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Invertebrate Pathology, 103(3) (2010) 170-178.

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Heat Shock Proteins and Inflammation, (2003) 43-54.

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domain, is involved in regulating melanocyte differentiation. J. Dermatol. Sci., 72(2013)

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246-251.

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[51] M. Bennett, N. Calakos, R. Scheller, Syntaxin: a synaptic protein implicated in docking

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of synaptic vesicles at presynaptic active zones. Science, 257(5067) (1992)255-259.

653

[52] S.H. Wong, T. Zhang, Y. Xu, V.N. Subramaniam, G. Griffiths, W. Hong, Endobrevin, a

654

novel synaptobrevin/vamp-like protein preferentially associated with the early endosome.

655

Molecular Biology of the Cell, 9(6) (1998) 1549-1563.

656

[53] R.B. Sutton, D. Fasshauer, R. Jahn, A.T. Brunger, Crystal structure of a SNARE complex

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involved in synaptic exocytosis at 2.4 Å resolution. Nature, 395 (1998) 347-353.

658

[54] A.L. Kolodkin, D.J. Matthes, C.S. Goodman, The semaphorin genes encode a family of

659

transmembrane and secreted growth cone guidance molecules. Cell, 75(7) (1994) 1389-1399.

660

[55] B. Marisa, O. Denis, C. Tito, C. Ernesto, "Chapter 4. Calcium in Health and Disease". In

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Astrid Sigel, Helmut Sigel and Roland K. O. Sigel (ed.). Interrelations between Essential

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Metal Ions and Human Diseases. Metal Ions in Life Sciences. 13(2013) 81–137.

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[56] B. Marisa, C. Tito, C. Denis, C. Ernesto, "Chapter 5 Intracellular Calcium Homeostasis

664

and Signaling". In Banci, Lucia (ed.). Metallomics and the Cell. Metal Ions in Life Sciences.

665

12(2013) 119–168.

666 667

668

Fig. 1. GO functional classification of the identified differentially expressed proteins in

669

hemocytes (A) and thoracic ganglion (B) based on biological processes, molecular function,

670

subcellular location.

671

Fig. 2. The subcellular location of differentially expression proteins identified in hemocytes

672

(A) and thoracic ganglion (B).

673

Fig. 3. KEGG analysis of differentially expressed proteins in hemocytes (A) and thoracic

674

ganglion (B).

675

Fig. 4. Proteins domain analysis of identified significantly changed proteins in hemocytes (A)

676

and thoracic ganglion (B) of E. sinensis after S. eriocheiris infection.

677

Fig. 5. Comparison of the expression profiles of selected significantly changed proteins in

678

hemocytes (A) and thoracic ganglion (B) as determined by qRT-PCR (black) and by TMT

679

analysis (gray).

680 681

Table 1 The primers used for qRT-PCR in this paper.

682

Table 2 Representative differently expression proteins in E. sinensis hemocytes with a

683

1.2-fold change post-injection with S. eriocheiris.

684

Table 3 Representative differently expression proteins in E. sinensis thoracic ganglion with a

685

1.2-fold change post-injection with S. eriocheiris.

686 687

Table S1 All the identified proteins in hemocytes of E. sinensis and corresponding information

688

analysis.

689

Table S2 All the identified proteins in thoracic ganglion of E. sinensis and corresponding

690

information analysis.

691

Table S3 Identified significantly changed proteins in hemocytes of E. sinensis and

692

corresponding information analysis.

693

Table S4 Identified significantly changed proteins in thoracic ganglion of E. sinensis and

694

corresponding information analysis.

695

Table S5 GO analysis of identified significantly changed proteins in hemocytes of E. sinensis

696

after S. eriocheiris infection.

697

Table S6 GO analysis of identified significantly changed proteins in thoracic ganglion of E.

698

sinensis after S. eriocheiris infection.

699

Table S7 KEGG analysis of identified significantly changed proteins in hemocytes of E.

700

sinensis after S. eriocheiris infection.

701

Table S8 KEGG analysis of identified significantly changed proteins in thoracic ganglion of E.

702

sinensis after S. eriocheiris infection.

703

Table S9 Proteins domain analysis of identified significantly changed proteins in hemocytes

704

of E. sinensis after S. eriocheiris infection.

705

Table S10 Proteins domain analysis of identified significantly changed proteins in thoracic

706

ganglion of E. sinensis after S. eriocheiris infection.

Table 1 The primers used for qRT-PCR in this paper. Name Sequence (5’-3’) Hemocytes Cathepsin L-qF AGCCAGTAGTCTGTGCCATCT Cathepsin L-qR CTGAGGACAAAGGTTTCGTAG CGATACAAGCCGACCTCACTA Hemocytin-qF Hemocytin-qR GATAGGCGACCAAACCACCAG Prx1-qF CGGTGGGGCAGACAAAAGTGA Prx1-qR AATGGGACAGCGGTGGTGGAC NOX5-qF TCACTATTTTGGCGAGGGCAT NOX5-qR TGGTCTAAGGGCAGGAAGGAA LGBP-qF GGGGTTGATGAGGTTGGTGGC LGBP-qR GGACTGGAGGGGCGAAGCGTT AGGGCAGATGCAATGGTGAGT Fer2-qF TGGGAATGAGATGCTGTGGAA Fer2-qR KPI-qF GGGCAGAATCTTGGGCACTCG KPI-qR GACGGCAACACCTACGGAAAC ACACCCCGTCCCGCAGACACTC VEGFR1-qF TCGGCTGGGCCTACCAAATCG VEGFR1-qR Thoracic ganglion SR-qF GGCTGATGCTTGGGTTTGGTC TCCGTTCCTGTTGTCGGGTGT SR-qR CTCCTCCTCCGAGAAGCCCAT ppA III-qF ACCCTGACTGCCAGCAAAACC ppA III-qR GCGGTGATGACGGGATTCTTA PPAF-qF TGCGTGCCCTACTACCTGTGC PPAF-qR AGGGCAGCGTGATGGGAATGT α2M-qF ACGGGGAGCCGAGCAATGAGT α2M-qR ACATCCCCGAGTGGCTGTTTG CCF-qF CCF-qR GCCTGTCCATCCCCTCCTTCA SNAP-25-qF TTCTCCTCCATCTCATCTTCA SNZP-25-qR TGCAACAAAACATCATCACAG GCATAATGCCCATTGCACCTC Rab10-qF GATGACGCCTTCAACACCACC Rab10-qR TGGCTCTGGACACGGGGATGA CD63-qF CGACGCTGAAGACCAGGACGC CD63-qR GAPDH-qF CTGCCCAAAACATCATCCCATC CTCTCATCCCCAGTGAAATCGC GAPDH-qR

Table 2 Representative differently expression proteins in change post-injection with S. eriocheiris. Protein name Accession Immunologic proteins NADPH oxidase 5(NOX5) dbj|BAL49600.1| trehalase ref|XP_008474901.1| thioredoxin reductase 1(Trx1) gb|AHJ86276.1| thioredoxin reductase 2(Trx2) ref|XP_002594765.1| peroxiredoxin 1(Prx1) gb|ACD68589.1| serine proteinase emb|CCW43203.1| melanization protease 1 emb|CCW43200.1| serine protease inhibitor gb|AAQ22771.1| mannose-binding protein(MBP) gb|ACR20475.1| hemocytin gb|ABG75717.1| cathepsin L gb|ABQ10739.1| cold shock domain-containing ref|NP_001246669.1| protein E1 granulins ref|XP_002415868.1| Glutathione S-transferase D7 gb|AGZ89666.1| (GST D7) thioredoxin-2 gb|ACQ59118.1| GILT-like protein 1 ref|XP_002126795.1| calcium-dependent secretion gb|KDR15617.1| activator calcium-activated chloride channel gb|EFX71591.1| regulator 1 crustacean calcium-binding protein sp|P80363.1| 23 calmodulin ref|XP_001948572.2| cathepsin B ref|XP_001658537.1| lysozyme C, milk isozyme gb|ADM33942.1| lectin gb|ADB10837.1| mannose receptor, C type ref|XP_002604716.1| lipopolysaccharide and gb|ACR56716.1| beta-1,3-glucan-binding protein (LGBP) Integrin alpha-PS3 ref|XP_002092640.1| vascular endothelial growth factor ref|XP_001356603.2| receptor 1(VGEFR1) serpin 3 gb|AHC06147.1| Kazal-type protease inhibitor (KPI) gb|AEW24506.1| clotting factor B2 gb|AFA42361.1| hemolymph clottable protein gb|ABW77320.1| ferritin2(Fer2) gb|ADF87491.1|

E. sinensis hemocytes with a 1.2-fold Score

Coverage Fold change

323.31 61.296 153.33 218.72 27.713 55.069 115.29 138.19 294.49 323.31 194.53 2.9886

29.2 17.2 45.8 47.2 34.3 15.1 37.1 33.7 70.9 49.3 37.4 1.6

1.28 1.28 1.465 1.459 1.382 1.282 1.458 1.524 2.261 1.31 1.324 1.202

20.658 21.467

5.9 17.6

0.748 0.622

70.101 308.24 24.581

35.2 40.5 2.5

0.681 0.716 0.709

26.141

27.7

0.506

168.03

46.1

0.564

3.9828 215.79 33.999 216.2 21.338 283.51

10.4 46.5 22.2 22.1 42.4 36.5

0.826 0.635 0.498 0.694 0.576 0.718

323.31 57.314

29.5 10.4

0.594 0.816

267.73 97.518 171.54 106.97 154.23

47.3 73.8 40.4 56.7 50.5

0.654 0.713 0.817 0.821 0.395

ferritin3(Fer3) alpha-2-macroglobulin domain-containing protein 8 Cytoskeleton proteins inverted formin-2 tubulin beta-1 chain myosin light chain alkali actin (Fragment) actin-A3b, cytoplasmic actin, alpha skeletal muscle troponin I troponin T tropomyosin Ras-like GTP-binding protein Rho (Rho)

gb|ACX30003.1| gb|ACX30003.1|

8.5583 139.77

7.6 27.9

0.536 0.754

ref|XP_785094.3| sp|Q25009.1| gb|AFP95338.1| gb|ABM74401.1| gb|AFC88033.1| gb|AAK84871.1| gb|AFW99837.1| ref|XP_002424248.1| gb|ABL89183.1| ref|XP_004519519.1|

27.386 323.31 75.366 15.462 9.7772 181.89 20.977 47.935 5.0249 26.141

10.8 67.2 31.2 51.9 64.1 48.2 22.3 18.7 26.2 27.7

1.444 1.292 0.554 0.604 0.745 0.589 0.494 0.64 0.561 0.506

Table 3 Representative differently expression proteins in E. sinensis thoracic ganglion with a 1.2-fold change post-injection with S. eriocheiris. Protein name Accession Score Coverage Fold change Immunologic proteins prophenoloxidase-activating factor gb|ACU65942.1| 417 28.1 1.45 prophenoloxidase activating enzyme gb|ABG67960.1| 82 10 1.32 III (ppAIII) kazal-type protease inhibitor gb|AEW24506.1| 103 46.9 1.454 pacifastin-like serine protease inhibitor gb|AFI56574.1| 290 1.971 36.3 LDLa domain-containing C-type lectin gb|AHB62786.1 98 16.7 3.617 beta-integrin gb|AFI56574.1| 244 11.5 1.533 integrin alpha 4 gb|AHH32887.1| 661 7.5 2.438 scavenger receptor (SR) emb|CUV67447.1| 56 1.8 2.014 cathepsin C gb|ABW74905.1| 200 17.1 1.364 lysosomal alpha-mannosidase-like ref|XP_013772922.1| 183 3.4 1.458 cathepsin D gb|AIF27797.1| 814 17.9 1.958 heat shock protein 21 gb|AET34915.1| 201 19.5 1.949 heat shock protein cognate 3 gb|AKB96213.1| 1176 31.2 1.212 heat shock protein 90 gb|AGC54636.1| 579 26.3 1.291 alpha-2-macroglobulin (α2M) gb|ADD71943.1| 568 15.2 1.254 alpha-2-macroglobulin 2(α2M-2) gb|ABM63360.1| 1356 16.4 2.252 hemocyanin subunit 6 gb|AEG64817.1| 2477 46.3 1.429 hemocytin 1 gb|KDR23192.1| 732 13.4 1.306 hemocytin 2 ref|XP_008552159.1| 1193 10.8 1.404 hemoglobin emb|CBN88274.1| 452 1.538 39.7 clotting protein precursor (CPP) gb|AAD16454.1| 4661 44 1.616 peroxiredoxin 6 gb|ACF35639.1| 228 38.6 1.243 catalase gb|ADD82543.1| 1402 49.6 1.242 cytochrome C dbj|BAJ22990.1| 101 9.1 1.279 peroxinectin gb|ADF87945.1| 598 16 1.282 transferrin gb|AEX92027.1| 2145 40.8 1.284 calreticulin gb|AEX92027.1| 941 38.9 1.222 Rab6-interacting family member 1 sp|Q99MI1.1| 424 11.3 0.709 Rab10 gb|AJC97117.1| 262 16.7 0.65 Rab6A ref|XP_012539045.1| 169 35.3 0.768 Rab-3 gb|AGC10823.1| 167 19.7 0.598 Rab27A-GAP-beta sp|Q4KMP7.3| 84 10.1 0.688 Ras-related protein gb|KFM66032.1| 377 38.6 0.773 G-protein coupled receptor Mth2-like ref|XP_013116211.1| 220 2.1 0.825 G protein beta 1 gb|AFD33363.1| 642 29.4 0.806 G-protein coupled receptor moody ref|XP_014257258.1| 74 2.2 0.616 catenin alpha ref|XP_008486042.1| 229 13.9 0.829 β-catenin gb|ACH92925.1| 137 7.4 0.661

coiled-coil domain-containing protein 6 mannose-6-phosphate isomerase (M6P) calpain B CD63 antigen (CD63) dLp/HDL-BGBP precursor collagenolytic serine protease protein laccase-4 C type lectin containing domain protein tetraspanin-1 chitinase 3 NADH dehydrogenase Nervous system related proteins calumenin EF hand Ca-binding protein sarco/endoplasmic reticulum calcium-ATPase astrocyte-derived neurotrophic factor extended synaptotagmin-2 syntaxin-8 LIM domain-binding protein tyrosine-protein kinase Src64B-like teneurin-3 neuronal calcium sensor 2 neurocalcin homolog calcium channel flower (CCF) calcium/calmodulin dependent kinase I plasma membrane calcium ATPase calcium-dependent secretion activator

gb|KFM66835.1| gb|KDR14999.1 gb|AAT77811.1| gb|AAT77811.1| gb|AHJ78589.1| gb|AEF33836.1| ref|XP_967238.1| gb|AEH05998.1| ref|XP_967238.1| gb|AKP18002.1| gb|KDR14969.1|

280 198 1118 41 637 129 53 513 62 69 59

14.4 19.7 22.9 7.2 5.5 11.4 4.7 25.8 8.8 3.2 22.2

0.743 0.598 0.796 0.8 0.806 0.346 0.542 0.791 0.811 0.393 0.767

gb|KDR16997.1|

361

22.2

1.649

prf||2104200A gb|AAW22143.1|

296 208

47.5 16.8

1.532 1.415

ref|XP_011149377.1| ref|XP_014262161.1| ref|XP_014225634.1| XP_012428257.1| ref|XP_009046870.1| gb|EGI63709.1| ref|XP_014468744.1| ref|XP_014218227.1| ref|XP_013192984.1| gb|AHG30903.1| gb|AFE19188.1| ref|XP_002431411.1|

100 195 61 202 93 242 970 435 114 170 580 247

13.5 9.9 12.1 11.5 8.6 2.5 45.2 50.3 14.8 18.1 13.9 5.1

2.744 2.348 1.213 1.943 2.609 1.298 0.683 0.689 0.69 0.826 0.815 0.741

calcium/calmodulin-dependent protein kinase type II (CaMK II) voltage-gated sodium channel synaptotagmin 1 semaphorin-1A synaptosome-associated protein of 25 kDa (SNAP25) synapse-associated protein of 47 kDa vesicle-associated membrane protein (VAMP) acetylcholinesterase 1 nicotinic acetylcholine receptor glutamate receptor glutamine synthetase 2 carnitine O-palmitoyltransferase 1

gb|ADX05543.1|

439

28.3

0.515

gb|ABL10360.2| ref|XP_011192446.1| ref|XP_014468222.1| dbj|BAC45225.1|

121 400 86 782

2.1 23.6 1.5 48.6

0.403 0.757 0.608 0.537

ref|XP_008200688.1| gb|AHG94982.1| gb|ACI16651.2| gb|AGK89910.1| ref|XP_013191738.1| gb|AFD52981.1| ref|XP_013773402.1| |ref|XP_013780285.1

229 420 160 884 149 320 71 176

27.2 30.8 13.1 21 5.6 0.764 2.7 17.8

0.688 0.56 0.793 0.689 0.696 26.8 0.77 0.739

Highlights

>TMT proteomic analysis help to understand the infection mechanism of S. eriocheiris at different stage with the different tissue (early stage with hemocyte and later stage with thoracic ganglion). >In total, 212 and 545 significantly different expression proteins identified in hemocyte and thoracic ganglion, respectively. >Some related pathways or proteins (proPO system, hemolymph coagulation system, nervous system development and signal transmission related proteins, etc.) were identified in hemocyte or thoracic ganglion of E. sinensis when the S. eriocheiris infection. >Some significantly changed proteins were identified and confirmed by qRT-PCR.