The function of serpin-2 from Eriocheir sinensis in Spiroplasma eriocheiris infection

Accepted Manuscript The function of serpin-2 from Eriocheir sinensis in Spiroplasma eriocheiris infection Meijun Yuan, Mingxiao Ning, Panpan Wei, Wenjing Hao, Yunting Jing, Wei Gu, Wen Wang, Qingguo Meng PII:

S1050-4648(18)30098-6

DOI:

10.1016/j.fsi.2018.02.036

Reference:

YFSIM 5143

To appear in:

Fish and Shellfish Immunology

Received Date: 18 December 2017 Revised Date:

12 February 2018

Accepted Date: 19 February 2018

Please cite this article as: Yuan M, Ning M, Wei P, Hao W, Jing Y, Gu W, Wang W, Meng Q, The function of serpin-2 from Eriocheir sinensis in Spiroplasma eriocheiris infection, Fish and Shellfish Immunology (2018), doi: 10.1016/j.fsi.2018.02.036. 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

The function of serpin-2 from Eriocheir sinensis in Spiroplasma

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eriocheiris infection

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Meijun Yuan a, 1, Mingxiao Ning a, 1, Panpan Wei a, Wenjing Hao a, Yunting Jing a, Wei

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Gu a, b, Wen Wang a, Qingguo Meng a, b,*

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a

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Laboratory for Aquatic Crustacean Diseases, College of Life Sciences, Nanjing

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Normal University, 1 Wenyuan Road, Nanjing 210023, China

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Jiangsu Key Laboratory for Biodiversity & Biotechnology and Jiangsu Key

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b

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Lianyungang, Jiangsu 222005, 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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*

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Tel: +86-25-85891955.

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E-mail address: [email protected].

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Corresponding authors: Qingguo Meng

ACCEPTED MANUSCRIPT Abstract

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Serpin families classified serine protease inhibitors regulate various physiological

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processes. However, there is not study on the role of serpin in immune responses

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against Spiroplasma eriocheiris as a novel causative pathogen in the Chinese mitten

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crab, Eriocheir sinensis. In our study, quantitative real-time PCR (qRT-PCR) revealed

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that the mRNA transcripts of Esserpin-2 were ubiquitous in every tissue, relative

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higher expression in hepatopancreas, gill and hemocytes, while the intestine, muscle,

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heart and nerve showed relative lower expression. Followed by infection with S.

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eriocheiris, the transcripts of Esserpin-2 were significantly down-regulated from 1 d

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to 7 d. After double-stranded RNA injection, the transcripts of Esserpin-2

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dramatically declined from 48 h to 96 h. The transcripts of proPO were found to be

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obviously increased after Esserpin-2 silenced, meanwhile, LGBP with no significant

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difference. The copy number of S. eriocheiris and subsequently the mortality of crabs

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in a silencing Esserpin-2 group were significantly less than control groups during

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infection. The subcellular localization experiment suggested that recombinant

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Esserpin-2 was mainly located in the cytoplasm. Finally, over-expression assay in

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Drosophila S2 cells indicated that Esserpin-2 could increase copies of S. eriocheiris

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and result in cell death. These findings demonstrated that Esserpin-2 involved in the

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innate immune mechanism of E. sinensis in response to S. eriocheiris infection.

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Keywords： ：Eriocheir sinensis, Esserpin-2, Spiroplasma eriocheiris

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1. Introduction

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ACCEPTED MANUSCRIPT The Chinese mitten crab, Eriocheir sinensis, is a commercial aquatic species in

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China [1]. With the increase of different diseases especially tremor disease (TD) [2]

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caused by Spiroplasma eriocheiris in recent years, the crab breeding has confronted

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serious production decline and economic losses. The S. eriocheiris was mainly seen in

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the hemocytes in the early processes of infection, and then transported to various

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tissues and organs by hemocytes [3]. Due to lack adaptive immunity, the crabs’ innate

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immune response played a vital role in protecting hosts from invading pathogens [4-5].

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Therefore, it was essential to research about innate immune-related genes of E.

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sinensis against S. eriocheiris to control the TD.

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In arthropods, the prophenoloxidase-activating system (proPO system) is a

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special and important innate immune defense mechanism and could involve in the

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immune response of Macrobrachium rosenbergii to S. eriocheiris challenge [6] .

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Upon injury or infection, microbial surface molecules such as lipopolysaccharide or

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peptidoglycan or β-1,3-glucan, initiate activation of a series of proteases, leading to

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the activation of zymogens of proteases, which in turn activate proPO to form melanin

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at the injury site or around invading organisms [7-8]. This system is mediated by

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several serine proteases and often regulated by irreversible protease inhibitors [9].

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Serine protease inhibitors, as an important protease superfamily including the Kazal,

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Kunitz, a-macroglobulin, and serpin families, have been widely identified and

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characterized biological functions [10]. Serpin families are found most proteins

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between 350 and 400 amino acids residues in length including a conserved structure

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with a reactive center loop near the C-terminus [11]. Except proPO inactivation,

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ACCEPTED MANUSCRIPT serpins can also participate in regulatory processes such as blood coagulation [12],

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cytokine activation [13] and antimicrobial peptides production. Indeed, the immune

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roles of serpins in crustaceans have been studied in several papers. Expression pattern

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of Fcserpin from Chinese shrimp Fenneropenaeus chinensis greatly fluctuated after

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challenged using white spot syndrome virus (WSSV), implying Fcserpin might have

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potential effects on the shrimp’s innate immunity against virus infection [14]. The

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expression levels of E. sinensis serpin (Esserpin) in hemocytes were significantly

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up-regulated after challenged by Vibrio anguillarum and Pichia pastoris, suggesting

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that the Esserpin was relevant to the crabs’ immune responses [15].

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However, no researching had been reported about the immune responses of

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serpins against S. eriocheiris, even though three serpin genes from E. sinensis, named

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Esserpin, Esserpin-2 and Esserpin-3 were cloned [15-16]. Here, Esserpin-2 immune

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roles during the stimulation by S. eriocheiris were explored. Therefore, the main

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objectives of this study were (1) to show a more in-depth understanding of the

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immune mechanism of Esserpin-2 protein, (2) to provide clues that Esserpin-2 protein

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might play potential functions against S. eriocheiris challenge.

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2. Materials and methods

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2.1. Animals, S. eriocheiris and Insect cell culture

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The E. sinensis (~ 25 g) were purchased from an aquaculture market in Nanjing,

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Jiangsu Province, China. Healthy E. sinensis were verified by Spiroplasma

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eriocheiris-negative results using hemolymph transmission electron microscope

ACCEPTED MANUSCRIPT (TEM) negative staining methods, and PCR of S. eriocheiris 16S rRNA [17]. The

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crabs were cultured at the laboratory (~ 26 ℃) in tanks containing aerated freshwater

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for 1 weeks before processing to allow acclimatization. S. eriocheiris were isolated

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from the crabs suffering TD using methods reported by Wang et al. [3] and cultured at

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30 ℃ with R2 medium. Drosophila Schneider 2 (S2) cells [18] were grown at 28 ℃ in

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Corning flasks in Schneider medium (Sigma, UK) supplemented with 10% fetal

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bovine serum (FBS) heat-inactivated at 56 ℃ for 30 min and antibiotics (100 U mL−1

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penicillin and 100 U mL−1 streptomycin).

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2.2. The spatiotemporal transcripts of Esserpin-2

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The hepatopancreas, intestine, heart, gill, muscle, nerve and hemocytes were

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collected from five untreated crabs to determine the tissue distribution of Esserpin-2

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transcripts. For the S. eriocheiris infection experiment, 100 healthy crabs were

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randomly divided into two groups including experimental group and control group.

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The challenge group were injected into 90 µL S. eriocheiris (108 cells/mL). In control

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group, the crabs were received an injection of 90 µL PBS. Five individuals hemocytes

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was collected at the time point of 0, 1, 3, 5, 7 and 9 d after challenged. The total RNA

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from the crab hemocytes was isolated by TRIzol Reagent (Invitrogen, USA) as

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described in the manufacturer’s protocol. RNA quality was measured by

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electrophoresis with 1% agarose gel. The total RNA was reverse-transcribed into

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cDNA using the PrimeScriptTM RT reagent Kit (Takara, Japan).

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Quantitative real-time PCR (qRT-PCR) was undergone with the reaction which

ACCEPTED MANUSCRIPT was added into a volume dose of 10 µL including 5 µL 2×SYBR Premix Ex TaqTM

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(Takara, Japan), 0.4 µL forward primer (10 µM, Table 1), 0.4 µL reverse primer (10

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µM, Table 1), 1µL cDNA template and 3.2 µL RNase-free Water. GAPDH as an

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internal control was amplified using primers (EsGAPDH-RT-F, EsGAPDH-RT-R)

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(Table 1). The PCR program was 95 ℃ for 30 s, followed by 40 cycles of 95 ℃ for 5 s

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and 60 ℃ for 30 s. The samples were repeated three times. All data were analyzed by

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2−∆∆CT method [19] and applied to relative mRNA expression levels as mean ± S.E..

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Statistical significance was analyzed by one-way analysis of variance (ANOVA)

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followed by Duncan and Tukey multiple comparison tests. Differences were

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considered to be significant at p < 0.05.

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2.3. Esserpin-2 RNA interference assay

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The dsRNA templates of Esserpin-2 and GFP (as control) were amplified using

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primer pairs (Esserpin-2-dsRNA-F and Esserpin-2-dsRNA-R; GFP-dsRNA-F and

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GFP-dsRNA-R) (Table 1). The dsRNAs of Esserpin-2 and GFP were synthesized with

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an in vitro transcription T7 kit (Takara, Japan). At the same time, agarose gel

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electrophoresis and quantified by spectrophotometry were utilized to monitor the

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synthetic quality of dsRNAs.

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To verify the efficiency of RNA interference (RNAi), the crabs were injected with

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60 µg Esserpin-2 dsRNA as the challenge group. Another group was injected with 60

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µg GFP dsRNA as the control. At 24 h post interference, the crabs were subjected to

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the same challenge to magnify the RNAi impact. After 48, 72 and 96 h of

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first-interference, the hemocytes of 5 crabs in each group were sampled. QRT-PCR

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were utilized to validate the knockdown of Esserpin-2 with primers Esserpin-2-RT-F/

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Esserpin-2-RT-R (Table 1). Meanwhile, the upstream and downstream genes of

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Esserpin-2, LGBP and proPO, were calculated by qRT-PCR. One hundred and eighty crabs were randomly divided into 6 groups including

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PBS group, PBS + S. eriocheiris group, GFP dsRNA group, GFP dsRNA + S.

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eriocheiris group, Esserpin-2 dsRNA group, and Esserpin-2 dsRNA + S. eriocheiris

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group, which contained 30 individuals, respectively. Esserpin-2 dsRNA (60 µg) were

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injected individually into Esserpin-2 dsRNA group and Esserpin-2 dsRNA + S.

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eriocheiris group. GFP dsRNA group and GFP dsRNA + S. eriocheiris group were

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injected with GFP dsRNA (60 µg), respectively. And the PBS group and PBS + S.

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eriocheiris group received individually an injection of PBS (60 µL). 24 h

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post-injection, the groups were challenged with the equal amount of dsRNAs or PBS.

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In addition, 90 µL S. eriocheiris (108 cells/mL) was inoculated into PBS + S.

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eriocheiris group, GFP dsRNA + S. eriocheiris group and Esserpin-2 dsRNA + S.

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eriocheiris group at 48 h post first-stimulation.

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Every five crabs from PBS + S. eriocheiris group, GFP dsRNA + S.eriocheiris

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group and Esserpin-2 dsRNA + S. eriocheiris group were sacrificed to detect the copy

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number of S. eriocheiris at 0, 1, 3, 5 and 7 d. The total DNA was extracted from

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hemocytes with Easy Pure Genomic DNA Kit (TransGen, China) and detected by

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absolute real-time PCR with primer pairs Se-QF and Se-QR [20] (Table 1). All

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experiments were performed in triplicate. Every day the cumulative mortality of the

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crabs was calculated to test the differences in mortality.

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2.4. Over-expression of Esserpin-2 analysis in Drosophila S2 cells A fusion plasmid pAc5.1-Esserpin-2-GFP which expressed the GFP-tagged

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Esserpin-2 protein was cloned [21-22] using the primer pairs Esserpin-2-F /

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Esserpin-2-R (Table 1) and the restriction endonuclease EcoR I and Apa I.

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Drosophila S2 cells were seeded onto the coverslips in three dishes to achieve

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60% confluence and 2 µg pAc5.1-Esserpin-2-GFP plasmid was transfected into these

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dishes with the FuGENE HD Transfection Reagent (Promega, USA). For subcellular

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localizations analysis after 48 h of transfection, the nucleus was stained with Hoechst

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33258 (Beyotime, China) and cell imaging was carried out to use a confocal laser

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scanning microscope (Nikon TI-E-A1R, Japan).

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Western blot assay was employed to demonstrate Esserpin-2 over-expression in

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Drosophila S2 cells. The experimental group were transfected with 4 µg

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pAc5.1-Esserpin-2-GFP plasmid (pAc5.1-GFP as the control group). 48 h later,

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Drosophila S2 cells were sampled and treated with Lysis buffer on ice [22]. Followed

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by sonication and centrifugation (13,000 × g, 15 min, 4 ℃), protein supernatants were

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subjected to concentration determination via bicinchoninic acid assay (BCA). The cell

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lysates were fractionated by 12% SDS-PAGE and then transferred onto a

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polyvinylidenefluoride (PVDF) membrane (Millipore, USA). The membrane was

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blocked with 5% BSA in TBST (TBS containing 0.05% Tween-20) at room

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temperature for 2 h, briefly washed with TBST, and incubated with anti-GFP (Trans,

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China) antibody (1:2000) overnight at 4 ℃. After washing, HRP-conjugated Goat

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Anti-Mouse IgG (TransGen, China) (1:5000) was incubated at room temperature for 2

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h and the brands were visualized with ECL (Vazyme, China). Furthermore, to determine the effects of Esserpin-2 over-expression on S.

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eriocheiris infection, 2 µg pAc5.1-Esserpin-2-GFP fusion plasmid was transfected into

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Drosophila S2 cells as the experimental group (S2 cells and S2 cells transfected with

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pAc5.1-GFP as the blank group and control group, respectively). S2 cells were

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stimulated by 200 µL S. eriocheiris (108 cells/mL) 24 h later. To examine S. eriocheiris

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copies, the S2 cells were sampled and carried out with absolute real-time PCR after

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48 h post infection.

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To verify cell viability, Drosophila S2 cells were cultivated into a 96-well plate

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with a final approximate 60% confluence. The methods of transfection and infection

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of the S2 cells were described as above. Drosophila S2 cells were imaged under an

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inverted fluorescence microscope (Nikon TI-S, Japan) after 48 h S. eriocheiris (10

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µL， 108 cells/mL) infection. EnoGene Cell Counting Kit-8 (CCK-8) (Beyotime, China)

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was performed to detect the viability of the S2 cells from 12 wells in every treatment.

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Ten µL CCK-8 was added into the cells and incubated for 4 h at 28 ℃ followed by

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measuring the absorbance at 450 nm via the microplate reader (Bio Tek, USA).

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3. Results

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3.1. The spatiotemporal transcripts of Esserpin-2

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The spatial distribution of Esserpin-2 was investigated using qRT-PCR and

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GAPDH as an internal control. As revealed by qRT-PCR, Esserpin-2 were ubiquitous

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in every tissue, relative higher expression in hepatopancreas, gill and hemocytes,

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while the intestine, muscle, heart and nerve showed relative lower expression (Fig.

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1A). The temporal transcripts profile of Esserpin-2 mRNA in the hemocytes of crabs

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were showed after stimulated by S. eriocheiris (Fig. 1B). In the whole course of

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infection, the transcripts of Esserpin-2 in the control group kept a steady level. For

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experimental group, the transcripts level of Esserpin-2 after S. eriocheiris injection

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began to have significant drop from 1 to 7 d (p < 0.05) and then no significant

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difference at 9 d.

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3.2. Esserpin-2 silencing and its regulation after S. eriocheiris stimulation

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To assess the functions of Esserpin-2 in the crab’s immune defense, Esserpin-2

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was interfered prior to S. eriocheiris injection and the transcripts levels of Esserpin-2

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mRNA was detected by qRT-PCR (Fig. 2A). The results showed that the transcripts of

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Esserpin-2 significantly declined in the experimental group compared with the control

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group (p < 0.05) and could last for 96 h after Esserpin-2 dsRNA inoculated. Therefore,

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the interference experiment of Esserpin-2 was efficient.

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In order to further find out the regulatory mechanism of Esserpin-2 gene in

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proPO system of the crabs, relative mRNA levels of LGBP (Fig. 2B) and proPO (Fig.

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2C) were determined using qRT-PCR. We found the levels of proPO, the downstream

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gene of Esserpin-2, were observably higher than the control groups (p < 0.05) from

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72 h to 96 h. But there is not significant changed in the levels of LGBP, the upstream

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gene of Esserpin-2.

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Absolute real-time PCR was used to measure the copy numbers of S. eriocheiris

ACCEPTED MANUSCRIPT in hemocytes (Fig. 3A). The data demonstrated that the numbers of S. eriocheiris in

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Esserpin-2 dsRNA +S. eriocheiris group remarkably declined compared with the

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control groups (GFP dsRNA + S. eriocheiris or PBS+ S. eriocheiris) from 3 to 7d (p <

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0.05). These results indicated that Esserpin-2 silencing could effectively reduce

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susceptibility of S. eriocheiris.

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The cumulative percent mortality of the crabs had a significant decline in

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Esserpin-2 dsRNA+S. eriocheiris group compared with the control groups (Fig. 3B).

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Briefly, the accumulative survival rate of the Esserpin-2 dsRNA+S. eriocheiris group

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was 70.67 % at 9 d, whereas GFP dsRNA+ S. eriocheiris group was 43.33 % and PBS

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+ S. eriocheiris group was 40.67 %, respectively. Together, these evidence clearly

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indicated that survival rate of the crabs was induced by limiting Esserpin-2 activity.

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3.3. Over-expression of Esserpin-2 in Drosophila S2 cells and its roles against S.

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eriocheiris infection

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To study the functions of Esserpin-2 in vitro, pAc5.1-Esserpin-2-GFP fusion

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plasmid was transfected into the S2 cells. The result show the GFP-fusion Esserpin-2

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protein mainly located in the cytoplasm (Fig. 4A). Furthermore, the expression of the

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Esserpin-2 protein was detected via western blot analysis. As indicated in Fig. 4B,

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Esserpin-2 fusion protein was captured in experimental group but did not present in

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the control group. Therefore, these results strongly showed that GFP-fusion Esserpin-2

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protein was emerged in S2 cells.

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In order to evaluate the immune functions of Esserpin-2 post S. eriocheiris

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infection, the pAc5.1-Esserpin-2-GFP plasmid was delivered into S2 cells then

ACCEPTED MANUSCRIPT stimulated with S. eriocheiris. Through absolute real-time PCR, the copy numbers of S.

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eriocheiris was assessed. By contrast, S. eriocheiris copies in the experimental group

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were more than the controls group (p < 0.05) (Fig. 5). The copy numbers of S.

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eriocheiris were 5248 copies/ng total DNA in experimental group at 48 h after infection,

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whereas 2422 copies/ng and 2403 copies/ng total DNA appeared in the control group

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and blank group, respectively. These data demonstrated that the over-expression of

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Esserpin-2 could increase invasion of S. eriocheiris into Drosophila S2 cells.

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The inverted fluorescence microscopy was employed to visualize the cellular

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morphology of Drosophila S2 cells. The proliferation ability of the S2 cells was

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reduced at 48 h after challenge in the experimental treatment by contrast with the

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control treatment and blank treatment (Fig. 6A, B, C and D). And the CCK-8 assay

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further revealed that S2 cell viability in the experimental group was noticeable less

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than that of the control group and blank group (p < 0.001) (Fig. 6E). All together, these

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results provided the first strong indication that the over-expression of Esserpin-2 was

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positively related to the numbers of S. eriocheiris entry into the S2 cells.

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4. Discussion

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With rapid development of intensive culture, TD which is caused by a novel

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pathogen, S. eriocheiris, is causing a serious damage to the aquaculture industry

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resulting in heavy economic loss [3]. In order to protect crabs from epidemic diseases,

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it is essential to attention about the innate immune system to defend against foreign

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pathogens. In invertebrates, it is universal that the serine protease cascades mediate

ACCEPTED MANUSCRIPT acute-phase regulation upon microbial challenge and these responses are often

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regulated by endogenous inhibitors including serpins to maintain homeostasis [23-25].

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More recent studies show serpins participate in the innate immune responses of

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invertebrates [26-27], but the immune functionality against S. eriocheiris remains

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obscure. Therefore, an Esserpin-2 gene was studied the functions in mediation E.

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sinensis immune response after S. eriocheiris challenge in this research.

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In this study, the Esserpin-2 could be detected in all the examined tissues of E.

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sinensis, with the higher expression levels in hepatopancreas, gill and hemocytes. This

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expression pattern in tissues was similar to Esserpin-3 in E. sinensis [16], but differed

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from Esserpin that the highest expression in gonad [15]. Considering the involvement

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of serpins in many immune processes [12-14] and their antimicrobial ability [11], the

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distribution of Esserpin-2 indicated that Esserpin-2 might play an important role in

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resistance to pathogenic infection. Besides, the wide expression of Esserpin-2

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transcripts might suggest the multiplex biological functions of Esserpin-2 in crabs.

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In crustaceans, hemocytes play extremely important functions in innate

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immunity system [28]. So, hemocytes was utilized as target organs to explore the

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responsiveness of Esserpin-2 expression after S. eriocheiris infection. The

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transcription of Esserpin-2 was remarkably decreased, which could show that the

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serpin relatively low expressed and relevant serine protease higher secreted during S.

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eriocheiris stimulation. This phenomenon might be caused by serine proteases

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involved in defense responses such as prophenoloxidase (proPO) activity and

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phagocytosis [29] in response to S. eriocheiris. The expression of Esserpin-2

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ACCEPTED MANUSCRIPT recovered until the end of the experiment, which might be related that Esserpin-2

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inhibited the over-expression of serine protease. This expression pattern was

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consistent with another antibacterial assay about Fcserpin [14].

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Previous researches suggested that pattern recognition proteins (PRPs), such as

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lipopolysaccharide and β-1,3-glucan binding protein (LGBP), had a striking function

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in recognition of invading species as foreign [30] and then triggered immune

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responses, including the activation of the prophenoloxidase system (proPO) by a

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cascade of serine proteases [31-32]. In our study, RNAi assay showed that the mRNA

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levels of proPO were continuously up-regulated based on the knockdown of

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Esserpin-2. This finding might reveal that Esserpin-2 acted as a negative regulator of

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the proPO activation. The similar result show Manduca sexta serpin-4 and serpin-5

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could suppress proPO activation [8]. Here we found that due to the silencing of

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Esserpin-2, the copies of S. eriocheiris and cumulative mortality of the crabs in

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Esserpin-2 dsRNA+S. eriocheiris group were both markedly decreased. This might

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suggest proPO system was activated after the knockdown of Esserpin-2 and resulted

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in increase of crab’s melanin, which could kill more invaded S. eriocheiris.

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Interestingly, serpin27A-deficient mutant from Drosophila were more susceptible

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than the wild-type after injection of bacteria or fungi [34]. Based on the observations

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from this study and from previous studies, it was speculated that S. eriocheiris entry

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might elicit LGBP, a pattern recognition receptor, to induce proPO activation cascade

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by inhibited multiple serpins such as Esserpin-2 against the invading organisms.

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ACCEPTED MANUSCRIPT To further investigate whether there was a similar function of Esserpin-2, the

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over-expression assay in Drosophila S2 cells was undertaken. Due to the lack of a

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complete crustacean hemocytes line, Drosophila S2 cells culture [18,22] was

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established as a good model to illuminate the functional study of Esserpin-2.

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Subcellular localization analysis demonstrated that Esserpin-2 was mainly identified

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in the cytoplasm. Apparently, the spatial distribution of specific serpins was crucial to

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their function in the immune response. Recently, several members of the serpin family

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have been found localized in the cell cytoplasm [16,35]. It can be deduced that the

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presence of Esserpin-2 in the cytoplasm, and even more close to the cell membrane,

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could be related to the signal transmission which inhibit the proPO activation system.

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Lately, we obtained additional evidence indicating that over-expression of Esserpin-2

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dramatically increased the copy numbers of S. eriocheiris and the cytotoxicity of

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Drosophila S2 cells. A parallel event was generated from Anopheles which was

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reported that serpin10 over-expression was accompanied by midgut cell death against

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Plasmodium infected [35]. In conclusion, several results demonstrated that Esserpin-2

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could regulated proPO activation in response to S. eriocheiris infection by inhibiting

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proPO activating system.

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In summary, a serpin (Esserpin-2) was involved in the immune functions of E.

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sinensis against S. eriocheiris stimulation. Moreover, Esserpin-2 mediated the

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immune responses by limiting proPO activation cascade. Therefore, this finding

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should provide a basis to pursue further precise functions and regulatory mechanism

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of Esserpin-2 and increase our knowledge of molecular events involved in the crabs,

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as well as invertebrates, immune responses.

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Acknowledgments

We thank Professor O. Roger Anderson (Columbia University) for editing the

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manuscript. The current study was supported by grants from the National Natural

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Sciences Foundation of China (NSFC Nos. 31570176; 31602198), the Natural

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Science Foundation of Jiangsu Province (Grant No. BK20151545), Project for

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Aquaculture in Jiangsu Province (Grant Nos. D2015-13; Y2016-28) and the project

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funded by the Priority Academic Program Development of Jiangsu Higher Education

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Institutions (PAPD).

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ACCEPTED MANUSCRIPT Figure Legends

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Fig. 1. Transcripts of Esserpin-2 mRNA in different tissues of healthy E. sinensis (A)

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and in E. sinensis hemocytes challenged by S. eriocheiris (B). The GAPDH was used

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as the reference gene. The assay was repeated three times. Vertical bars represented

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the mean ± S.E. (n = 15). The asterisks (*) indicated significant differences (p < 0.05)

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compared with values of the control.

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Fig. 2. The transcripts analyses resulted from the Esserpin-2 dsRNA interference. The

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transcripts level of (A), Esserpin-2; (B), LGBP and (C), proPO were detected at 48,

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72 and 96 h post dsRNAs injection to detect the effects of gene silencing. GAPDH

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was used as reference gene. Vertical bars represented the mean ± S.E. (n = 15).

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Significant differences (p < 0.05) were indicated by asterisks (*).

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Fig. 3. The changes of S. eriocheiris copies in E. sinensis hemocytes (A) and survival

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rate of the crabs (B) post challenge. Absolute real-time PCR analysis was performed

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in triplicate for each sample. The asterisks (*) indicated significant differences (p <

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0.05).

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Fig. 4. Subcellular localization and over-expression of Esserpin-2 in Drosophila S2

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cells. A, the cells were visualized by a confocal laser scanning microscope. (1),

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ordinary light with the microscopy visualized the cells. (2), cell nucleus was stained

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by Hochest 33258 (blue). (3), S2 cells transfected with pAc5.1-Esserpin-2-GFP

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(green). B, the protein expression levels of Esserpin-2 in the S2 cells were analyzed

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by Western blotting with an anti-GFP antibody. (1), experimental group; (2), control

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group; (M), protein marker. Approximate molecular sizes: GFP-Esserpin-2, ~77 kDa;

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Fig. 5. The effect of Esserpin-2 over-expression in Drosophila S2 cells in response to

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S. eriocheiris infection. Absolute real-time PCR was conducted to detect the copy

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number of S. eriocheiris in the S2 cells. The data were repeated three times. Vertical

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bars represented the mean ± S.E. (n = 3). Statistical significance was indicated: *p <

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0.05.

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Fig. 6. The cytotoxicity of Esserpin-2 over-expression in Drosophila S2 cells under

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stimulation by S. eriocheiris. (A, B, C, D) represented the S. eriocheiris-free group, S.

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eriocheiris only group, S. eriocheiris + GFP group and S. eriocheiris +

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GFP-Esserpin-2 group, respectively. Bar = 100 µm. (E) the cell viability was

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conducted by CCK-8. Cells used for different treatments were shown on the abscissa,

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and the rate of cell viability on the ordinate. The data were presented as the mean ±

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S.E. (n = 36) from three independent experiments. Significant difference was

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indicated: ***p < 0.001.

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ACCEPTED MANUSCRIPT Table 1 Primers used for real-time quantitative and cloning analyses of Esserpin-2 Sequence(5’-3’)

Esserpin-2-RT-F

GTTCAAGAAGTCTGCCACCG

Esserpin-2-RT-R

TGTACATCGAAACAGCCTCCC

EsGAPDH-RT-F

CTGCCCAAAACATCATCCCATC

EsGAPDH-RT-R

CTCTCATCCCCAGTGAAATCGC

Esserpin-2-dsRNA-F

GCGTAATACGACTCACTATAGGCGGCCCCATCCTGCACCACGC

Esserpin-2-dsRNA-R

GCGTAATACGACTCACTATAGGCACTACAGGCTGTCTACGAGG

GFP-dsRNA-F

GCGTAATACGACTCACTATAGGTGGTCCCAATTCTCGTGGAAC

GFP-dsRNA-R

GCGTAATACGACTCACTATAGGCTTGAAGTTGACCTTGATGCC

LGBP-RT-F

TCATCAAGCCGCAACTCAC

LGBP-RT-R

TCCGAAGCCTGGCACTCA

ProPO-RT-F

GTGAAGGCAAGCGGGTGA

ProPO-RT-R

CCCTGTGAGCGTTGTCCG

Se-QF

CGCAGACGGTTTAGCAAGTTTGGG

Se-QR

AGCACCGAACTTAGTCCGACAC

Esserpin-2-F

TAGTCCAGTGTGGTGGAATTCAAAATGGACACCCGAACATCATG

Esserpin-2-R

AGGCTTACCTTCGAAGGGCCCCGAGGCCTTGGGATTCTTG

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Highlights > Esserpin-2 involved in the immune responses of E. sinensis to an S. eriocheiris challenge.

decrease in Esserpin-2 RNAi assay.

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> The copies of S. eriocheiris and crabs’ death rate were obviously

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viability in over-expression assay.

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> Esserpin-2 could increase copies of S. eriocheiris and decrease of cell