SpBOK inhibits WSSV infection by regulating the apoptotic pathway in mud crab (Scylla paramamosain)

Journal Pre-proof SpBOK inhibits WSSV infection by regulating the apoptotic pathway in mud crab (Scylla paramamosain) Shanmeng Lin, Yuyong He, Yi Gong, Yueling Zhang, Hongyu Ma, Huaiping Zheng, Shengkang Li PII:

S0145-305X(19)30543-9

DOI:

https://doi.org/10.1016/j.dci.2019.103603

Reference:

DCI 103603

To appear in:

Developmental and Comparative Immunology

Received Date: 7 November 2019 Revised Date:

27 December 2019

Accepted Date: 28 December 2019

Please cite this article as: Lin, S., He, Y., Gong, Y., Zhang, Y., Ma, H., Zheng, H., Li, S., SpBOK inhibits WSSV infection by regulating the apoptotic pathway in mud crab (Scylla paramamosain), Developmental and Comparative Immunology (2020), doi: https://doi.org/10.1016/j.dci.2019.103603. 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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SpBOK inhibits WSSV infection by regulating the apoptotic

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pathway in mud crab (Scylla paramamosain)

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Shanmeng Lina,b,c,1, Yuyong Hea,b,c,1, Yi Gonga,b,c, Yueling Zhanga,b,c, Hongyu Maa,b,c, Huaiping

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Zhenga,b,c, Shengkang Lia,b,c*

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a

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China

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b

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c

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Guangdong Provincial Key Laboratory of Marine Biology, Shantou University, Shantou 515063,

Marine Biology Institute, Shantou University, Shantou 515063, China

STU-UMT Joint Shellfish Research Laboratory, Shantou University, Shantou 515063, China

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Correspondence author: Shengkang Li. Tel: +86-754-86502485. Fax: +86-754-86503473.

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E-mail: [email protected].

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1

These authors contributed equally to this paper.

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Running title: SpBOK inhibits WSSV infection via apoptotsis

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Abstract

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B-cell lymphoma 2 (Bcl-2) related ovarian killer (BOK) is a member of the Bcl-2 family,

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which has a similar function to BAX and BAK in the process of apoptosis. However, how BOK

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activates the intrinsic (mitochondrial) apoptotic pathway remains poorly understood in

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invertebrates. In this study, SpBOK identified in mud crab is an important effector responsible for

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the anti-WSSV (White Spot Syndrome Virus) infection by activating the apoptotic pathway. The

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SpBOK gene encoded a 282 amino acid peptides (molecular mass of 29 kD), which contained four

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distinct Bcl-2 family homology (BH) domains. SpBOK was widely expressed in all tested tissues

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and up-regulated after WSSV infection in vivo. The role of SpBOK on the anti-WSSV response in

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mud crab was investigated by using the RNAi approach in vivo. SpBOK exerted a regulatory role

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in changing the mitochondrial membrane potential (⊿ψm) and activating the caspase signaling

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and thus induced apoptosis. Moreover, the results showed that WSSV replication in mud crab

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could be effectively inhibited by SpBOK. Therefore, the results of this study demonstrated that

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SpBOK can inhibit WSSV infection by regulating the intrinsic apoptosis pathway in mud crab.

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Keywords: BOK; mitochondrial membrane potential (⊿ψm); apoptosis; White spot syndrome

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virus (WSSV)

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Introdution

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Apoptosis is an important mechanism that is required for the normal development of cells

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and the maintenance of tissue homeostasis in multicellular animals by eliminating superfluous or

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potentially harmful cells (Hengartner, 2000). Mis-regulated apoptosis can induce various diseases,

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including cancers, developmental disorders, autoimmune and neurodegenerative diseases

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(Desagher and Martinou, 2000; Yaron and Hermann, 2011). In vertebrates, shaping embryos and

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maintaining homeostasis in adult tissues are intimately associated with the apoptosis (Hipfner and

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Cohen,2004). The apoptosis process is related to characteristic cell changes, including cell

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membrane blebbing, cell shrinkage, DNA fragmentation, apoptotic body formation, and

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engulfment by phagocytes, which thereby prevents inappropriate inflammation in tissues (Adams

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and Cory, 2018). In principle, apoptosis occurs mainly through two main intrinsic and extrinsic

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pathways. The extrinsic pathway is stimulated by various death-inducing ligands, such as tumor

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necrosis factor (TNF), TNF-related apoptosis-inducing ligand (TRAIL) and fatty acid synthetase

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ligand (FasL). The binding of ligands to death receptors induces the formation of a death-inducing

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signaling complex (DISC), which activates the downstream caspase cascade (Long and Ryan,

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2012). The intrinsic pathway is mainly regulated by the mitochondrial and a variety of

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intracellular stimuli. The stimuli can activate the pro-apoptotic members of the B cell lymphoma

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protein (Bcl-2) family and stimulate the mitochondrial membrane potential (⊿ψm). Then the

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cytochrome c together with other proteins can release from the mitochondrial outer membrane,

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leading to the activation of caspase-3 and finally inducing the apoptosis (Shimizu et al. , 2000).

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The intrinsic pathway is involved in regulatory elements known as Bcl-2 family proteins,

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which are classified into pro-apoptotic and pro-survival proteins, respectively. The pro-apoptotic

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proteins are composed of BAK, BAX, BOK, and BH3-only proteins. The BAK, BAX, and BOK

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proteins are responsible for the permeabilization of MOMP, while the BH3-only proteins

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(including BIM, BID, PUMA, BIK, BAD, BMF, NOXA, and HRK) regulate the intrinsic

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apoptotic pathway. The pro-survival proteins consist of Bcl-2, Bcl-xL, Mcl-1, Bcl-B, Bcl-w, A1

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and BAG proteins (Ke et al., 2015; Kvansakul et al., 2017; Waseem Ahmad et al., 2015). Bcl-2

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ovarian killer (BOK), a pro-apoptotic protein, has been reported for the first time in a yeast

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two-hybrid screen (Ke et al., 2015), which showed 70–80% sequence homology to BAK and BAX

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(Carpio et al., 2015a). However, unlike BAX and BAK proteins, BOK cannot be inhibited by

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anti-apoptotic proteins, i.e. Bcl-2, Bcl-xl and Mcl-1 (Llambi et al., 2016) and plays an important

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role in regulating the apoptotic response to endoplasmic reticulum (ER) stress, tumor suppress,

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neuronal injury and inhibiting the virus infection in fish cell (Cai et al., 2016; D'Orsi et al., 2016;

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Zheng et al., 2018). BOK can target the membranes of ER, Golgi, nucleus and mitochondrial

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through its C-terminal transmembrane domain (Echeverry et al., 2013; Onyeagucha et al., 2017).

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Caspases are a family of cysteine proteases that play an important role in programmed cell

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death (including apoptosis) (Los et al., 2001; Thornberry and Lazebnik, 1998). Caspase-3 is one of

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the most crucial effectors which lead to proteolysis of protein substrates and protect living cells

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from apoptosis (Fan et al., 2010; Janicke and U., 1998; Salvesen and Dixit, 1997; Schlegel et al.,

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1996; Thornberry and Lazebnik, 1998).

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White spot syndrome virus (WSSV), a major devastating pathogen affecting shrimp farming,

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was first reported in Taiwan in the 1990s. The viral infection can induce 100% accumulative

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mortality of shrimp in 2-10 days (Wu et al., 2005; Xu et al., 2007). In addition to shrimp, other

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crustaceans, i.e. crabs and crayfish, has been also infected by the virus (WSSV) (Wu et al., 2005).

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Previous studies have reported that apoptosis could be found in the hemocyte cells of the mud crab

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during WSSV infection (Flegel et al., 1999; Ma et al., 2019; Chen et al., 2019). In this study,

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SpBOK was cloned and characterized. The results of this study showed that SpBOK could inhibit

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WSSV replication by activating the intrinsic apoptotic pathway of the hemocytes in mud crab

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Scylla paramamosain.

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2. Materials and methods

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2.1. Crab culture and WSSV challenge

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Healthy crabs (average weight 35 g) were acclimatized in the tanks under laboratory

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conditions (water salinity of 10‰ and temperature of 25 oC) for three days. Before the challenge

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experiment, the crabs were randomly selected for the detection of WSSV copies using

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WSSV-specific primers and the TaqMan probes (Table 1) to ensure that the crabs were virus-free.

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In the infection experiment, the crabs were divided into two groups of treatment and control (three

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crabs for each). Each crab of treatment and control groups was injected with either 200 µL of

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WSSV (106 copies/mL in PBS) and PBS only, respectively. At 0, 24, 48 and 72 hpi, the hemocytes

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or tissues of crabs were sampled.

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2.2. Gene cloning

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The open reading frame (ORF) sequence of SpBOK was obtained by transcriptomic

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sequencing. The partial sequence of SpBOK was amplified by RT-PCR using the corresponding

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primer (Table 1).

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2.3. Bioinformatics analysis

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The amino acid sequence of SpBOK (GenBank accession no. MK779314) and the similarity

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analysis was conducted using the NCBI blast program . Domain architecture of the SpBOK

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protein was performed using the SMART software (http://smart.embl-heidelberg.de/) and BLAST

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program (http://www.ncbi.nlm.nih.gov/blast/). Homology modeling of SpBOK was predicted by

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SWISS-MODEL (http:// swissmodel. expasy. org/). DNAMAN (Anja et al., 2006) was used for

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multiple alignments of amino acid sequences, and MEGA 5.2 (Luo et al., 2016) was used for

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constructing the phylogenetic tree.

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2.4. Antibody preparation of SpBOK

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The ORF sequence of SpBOK was expressed in Escherichia coli. The gene was amplified

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from crab hemocytes with the corresponding primers (Table 1). The PCR procedure was as

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follows: one cycle at 95 oC for 3 min; 40 cycles at 95 oC for 30 s, 57.9 oC for 30 s, and 72 oC for 1

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min; and one cycle at 72 oC for 10 min. The product was then cloned into pGEX6P-1 vectors. The

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recombinant protein was purified by affinity chromatography with GST-resin (Transgen biotech,

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Beijing). SpBOK antiserum preparation was performed as previously described (Du et al., 2007).

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2.5. Western blot analysis

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Tissue proteins were obtained from the hemocytes of the treatment and control groups. The

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proteins were homogenized with RIPA lysis buffer (Beyotime, China) following the

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manufacturer’s instructions. The samples were separated by 10.0% SDS-polyacrylamide gel

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electrophoresis and then transferred onto nitrocellulose membranes. The membranes were blocked

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with 5% non-fat milk in TBST (20 mM Tris-HCl, 150 Mm NaCl, 0.05% (v/v) Tween 20, pH 8.0)

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for 1 hpi at room temperature and incubated with 1/500 diluted antiserum against SpBOK for 14

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hpi at 4 oC. Then the membranes were incubated with goat anti-rabbit IgG (1/5000 diluted in

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TBST) for 2 hpi at room temperature. The membranes were detected by Western Lightning®

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Plus-ECL (Perkin elmer, USA) after rinsing with TBST for 20 min.

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2.6. RNA interference

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RNA interference (RNAi) assay was performed to knockdown the expression of SpBOK in

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mud crabs. The siRNAs were amplified by the primers (Table 1). The in vitro Transcription T7 kit

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(Takara, Japan) was used to synthesize the siRNA following the manufacturer’s instructions.

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siRNAs (25 µg) for BOK was injected into the crab and siGFP was used as a control. Twelve

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hours after injection, siRNAs (25 µg) were used to inject into crabs for the second time. The

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hemocytes were collected from the crabs at 24 hours post-injection (hpi) and total RNA was

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extracted using TRIzol reagent (Cwbio, Beijing, China). The efficacy of the gene knockdown was

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assessed by qRT-PCR using the primers RT-BOK-F and RT-BOK-R (Table 1). The protein was

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also collected to analyze the efficacy of the RNAi by western blotting. To analyze the role of

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SpBOK in regulating the anti-WSSV pathway, the crabs were divided into three groups: two RNAi

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groups were challenged with WSSV (106 copies/crab) and the remaining group was received PBS

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injection only as controls. At 0, 24 and 48 hpi, the hemocytes of three crabs from each group were

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collected and used for detecting the WSSV copy number using qRT-RCR, the⊿ψm, and the

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apoptotic rate.

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2.7. Quantification of WSSV copies with qRT-PCR

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At different time points after WSSV challenges, the muscle of crabs was collected and used

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for extracting the genomic DNA using TIANamp Genomic DNA Kit (Tiangen, China). The

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extracted DNAs were used as templates in the qRT-PCR using WSSV-specific primers and

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TaqMan probes. The qRT-PCR procedure was one cycle of 95 oC for 1 min, 40 cycles of 95 oC for

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30 s, 52 oC of 30 s, 72 oC for 30 s.

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2.8. Flow cytometric analysis

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Apoptosis rate was observed by flow cytometry using the FITC Annexin V Apoptosis

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Detection Kit l (BD PharmingenTM, US ). After centrifugation at 600 ×g at 4 oC for 10 min, the

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hemocytes were collected, rinsed twice with 1 X PBS and then centrifuged at 550 ×g at 4 oC for 5

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min. The cells were stained in 100 µL of 1× Binding buffer, followed by 5 µL of PI and 5 µL of

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FITC Annexin V. After incubation for 15 min at RT (25 oC) in dark, the cells were resuspended in

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400 µL of 1× Binding buffer and the apoptotic rate of the cells was analyzed using a flow

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cytometry.

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2.9. Determination of mitochondrial membrane potential (⊿ ⊿ψm).

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The mitochondrial membrane potential assay kit with JC-1 (Beyotime, China) was used to

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measure the mitochondrial membrane potential according to the manufacturer’s protocols. The

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hemocytes of crabs were collected by centrifugation at 600 ×g at 4 oC for 5 min. The pellet was

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resuspended in 1× PBS solution and the suspension was centrifuged at 550 ×g at 4 oC for 5 min.

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The cells were stained with 0.5 mL of JC-1 staining solution at 37 oC for 30 min in a 1.5 mL EP

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tube and then rinsed twice with JC-1 dying buffer. The fluorescence intensity was measured at the

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excitation wavelength of 530 and 590 nm in a Microplate Reader, respectively, and the same

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samples were observed by a confocal microscope (ZEISS, Germany).

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2.10. Activation of caspase-3

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The Caspase 3 Activity Assay Kit (Beyotime, China) was used to measure the caspase 3

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activity according to the manufacturer’s protocols. Cells were collected by centrifugation at 600

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×g at 4 oC for 10 min and wash with 1× PBS buffer at 600 ×g at 4 oC for 5 min. The cells were

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resuspended in the lysis buffer for 15 min and the supernatant was obtained after centrifugation at

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16000 ×g at 4 oC for 15 min. Each sample was incubated with 40 µL of detection buffer and 10 µL

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of Ac-DEVD-PNA (2 mM) of each substrate at 37 oC for 120 min. The samples were

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colorimetrically monitored at 405 nm in a Microplate Reader.

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3. Results

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3.1. Molecular characteristic of SpBOK

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The ORF sequence of SpBOK comprises 849 nucleotide bases in length, encoding 282 amino

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acid residues (molecular weight of 29 kD). Multiple sequence alignment showed a high amino

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acid sequence homology between SpBOK and BOK from other species, such as Palaemon

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carinicauda (84.03%), Penaeus vannamei (83.27%), and Macrobrachium rosenbergii (88.35%)

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(Fig. 1A). A phylogenetic tree was constructed to assess the evolutionary relationship between

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SpBOK and other known BOKs using the neighbor-joining method with 1000 replicates. As

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expected, the SpBOK was closely related to that from P. carinicauda and M. rosenbergii (Fig. 1B).

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The putative SpBOK protein contains BH1, BH2, BH3, BH4, and TM domains (Fig. 1C), which

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was also shown in the 3D structure of SpBOK (Fig. 1D).

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3.2. SpBOK responses to the WSSV infection

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qRT-PCR was used to examine the mRNA expression of SpBOK in various tissues (intestine,

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skin, heart, hepatopancreas, muscle, gills, and hemocytes) of healthy mud crabs (Fig. 2A). As

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shown in Fig. 2A, SpBOK was more abundant in the intestine and skin than that in the heart,

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hepatopancreas, muscle, gill, and hemocytes; the transcription of SpBOK was lower in the

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hepatopancreas and hemocytes than in the other tissues (Fig. 2A). The WSSV copies increased

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gradually from 0 hpi to 72 hpi in crabs infected with WSSV (Fig. 2B). The expression of SpBOK

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in the hemocytes of crabs infected with WSSV was analyzed by qRT-PCR. Compared with the

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controls, the mRNA level of SpBOK was significantly upregulated at 48 and 72 hpi (Fig. 2C) and

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the translational level was also significantly upregulated at 24 and 72 hpi (Fig. 2D). These results

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indicated that the expression of SpBOK was highly induced by WSSV infection.

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3.3. Apoptosis of hemocytes induced by WSSV infection

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To investigate whether WSSV infection could activate the anti-virus apoptosis pathway, mud

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crabs were challenged with WSSV and the apoptotic rate of hemocytes (at 0, 24, 48 hpi with

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WSSV) was evaluated by Annexin V/PI staining. The results revealed that the apoptotic rate of

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hemocytes was significantly increased at 24 and 48 hpi compared with the controls (Fig. 3 A, B).

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The data suggested that the apoptosis of hemocytes could be activated in vivo in response to the

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viral infection.

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3.4. The role of SpBOK in antiviral immunity

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To confirm that SpBOK is involved in the antiviral response, SpBOK was silenced by

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gene-specific siRNA knockdown technique. The qRT-PCR analysis revealed that SpBOK mRNA

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in hemocytes was significantly inhibited at 24 hpi with siBOK (Fig. 4A and 4B). To investigate

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whether the expression of SpBOK could suppress WSSV replication, PBS only or WSSV was

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injected into the siBOK-injected and siGFP-injected groups, respectively. The results showed that

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the WSSV copies in the SpBOK-silenced crabs were significantly increased compared with the

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controls (Fig. 4C). Taken together, the results suggested that SpBOK played an important role in

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inhibiting WSSV replication.

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3.5 SpBOK regulates mitochondrial membrane potential (⊿ ⊿ψm )

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The decrease of mitochondrial membrane potential (⊿ψm) is an important event in the

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apoptosis process. To gain further insight into the role of SpBOK in mediating ⊿ψm in mud

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crabs, the SpBOK gene was firstly knockdown by SpBOK-specific siRNA injection and followed

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by WSSV challenge. The results revealed that the samples of siBOK-injected crabs (at 48 hpi with

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WSSV) had a significantly weakened red fluorescence signal and strengthened green fluorescence

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signal, compared with the PBS controls, or an opposite trend of fluorescence signals compared

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with that of siGFP-injected crabs and WSSV-injected crabs (Fig. 5A). In addition, the

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mitochondrial⊿ψm of siBOK-injected crabs challenged with WSSV was significantly reduced

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compared with the PBS controls, while the mitochondrial⊿ψm of those was markedly increased

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(P<0.01) compared with the siGFP-injected groups (Fig. 5B).

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3.6 SpBOK mediates the activation of apoptosis

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Apoptotic induction was analyzed by flow cytometry staining with Annexin V/PI. The results

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showed that the siBOK-injected crabs could efficiently increase the apoptosis rate at 48 hpi with

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WSSV. On the other hand, the apoptotic rate in the siBOK-injected crabs were slightly reduced

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compared with the siGFP-injected crabs or the WSSV-injected crabs at 48 hpi with WSSV (Fig. 6

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A and 6B). The caspase-3 activity was found to significantly decrease in the siBOK-injected crabs

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challenged with WSSV compared with the controls (Fig. 6C). Thus, it is indicated that SpBOK

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could suppress the WSSV infection by activating the intrinsic apoptotic pathway directly.

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Discussion

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In this study, SpBOK was identified for the first time in the mud crabs, which is a

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pro-apoptotic member of the Bcl-2 family, playing an important role in the antiviral immune

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response. The SpBOK could induce the cell apoptosis pathway by decreasing the ⊿ψm to

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suppress the WSSV infection. SpBOK mRNA was found to be expressed in the intestine with the

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most abundant distribution, followed by the skin, gill, heart, hemocytes, muscle, and

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hepatopancreas. This is similar to the case of that in mammals, where BOK has been detected to

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widely express in different organs, including the brain, gastrointestinal, lung (Moravcikova et al.,

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2017), liver cancer cells (Li et al., 2013), neuronal cell, and mouse embryonic fibroblasts (MEFs).

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Notably, in the cancer cells, BOK has been reported to play a key role in regulating ER

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stress-induced apoptosis (Sopha et al., 2017). In the neuronal cells, BOK regulates the Ca2+

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homeostasis in neurons (D'Orsi et al., 2017) and the trophoblast cell proliferation with

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non-apoptotic function (Ray et al., 2010), and even promotes survival rather than death (D'Orsi et

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al., 2016; Echeverry et al., 2013). The MEFs which lacking BOK can resist the ER stress-induced

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apoptosis (Carpio et al., 2015b). Furthermore, previous studies have found that BOK is mostly

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expressed in the kidney and spleen of fish, the ovary of freshwater prawn, and other tissues (Cai et

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al., 2016; Chaurasia et al., 2015). Thus, these results revealed that BOK plays multiple roles in

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different tissues.

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BOK contains a highly conserved multi-domain of Bcl-2 family and shares high homology

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with the pro-apoptotic proteins BAK and BAX. BOK also has the function of pro-apoptosis

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(Einsele-Scholz et al., 2016). In mammals, BOK can promote mitochondrial apoptosis to

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response to the ER stress induced by UV, etoposide, staurosporine and other stimuli (Carpio et al.,

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2015a). BOK has been found to express in a variety of tissues of grouper (Epinephelus coioides)

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after challenge with Singapore grouper iridovirus (SGIV) and the overexpressed BOK can

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significantly decrease the SGIV replication (Cai et al., 2016). In freshwater prawn (M.

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rosenbergii), the challenge with viruses (MSBV and MrNV) or bacteria (Aeromonas hydrophila

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and Vibrio harveyi) could initiate the apoptotic process via the BOK-dependent apoptotic

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signaling pathway (Chaurasia et al., 2015). In this study, the expression of SpBOK in the

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hemocytes of crabs following the WSSV challenge was investigated. The result showed that the

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mRNA level and the protein level of SpBOK were significantly increased at 24 and 72 hpi,

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indicating the important role of SpBOK in response to WSSV infection. The results of the

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hemocyte apoptotic rate by Annexin V/PI staining showed that WSSV infection induces the cell

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apoptosis and the replication of WSSV was significantly increased in BOK-silenced crabs. These

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results indicated that SpBOK could inhibit WSSV replication in mud crabs.

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Changes in mitochondrial membrane potential (⊿ψm) were considered to be an early event

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in the intrinsic apoptosis pathway (Austin et al., 2008). Previous reports in mammals suggested

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that viral replication leads to cellular responses, including ER stress (He, 2006). BOK serves as a

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sensor for ERDA dysfunction during ER stress, which triggers the mitochondrial membrane

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permeabilization in the absence of BAK and BAX. The changes of ⊿ψm promote the open of

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permeability transition pores and the release of inter-membrane space protein, which induce the

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apoptosis (He et al., 2017). In several mammalian cell models, the overexpression of BOK can

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promote mitochondrial outer membrane permeabilization (MOMP) and apoptosis (Brem and Letai,

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2016; D'Orsi et al., 2017). While in some studies reported that disruption of the mitochondrial

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outer membrane is not enough to release cytochrome c completely and the loss of membrane

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potential (⊿ψm) faclitate cytochrome apoptogenic factors release and apoptosis(Gottlieb et al.,

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2003). Thus, our data suggested that SpBOK could induce the loss of ⊿ψm and these might

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activate the apoptosis.

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In addition, the apoptotic rate was observed and the data showed that the apoptotic rate was

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significantly decreased in siBOK-injected crabs compared with the controls. Furthermore, the data

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of caspase-3 activity indicated that the relative activity significantly decreased compared with the

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controls, suggesting that SpBOK might promote apoptosis of the hemocytes in mud crab. The

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results herein showed a similarity to that reported previously (Carpio et al., 2015). Taken together,

278

these results indicated that SpBOK could induce the hemocyte apoptosis in mud crab after WSSV

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infection.

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In conclusion, SpBOK was identified from mud crab. The apoptosis would be induced and

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the upregulation of SpBOK was observed in the hemocytes of mud crab upon the WSSV challenge.

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SpBOK can effectively inhibit WSSV replication and induce MOMP. This directly activates the

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downstream caspase signaling and stimulates the apoptosis of the hemocyte in mud crab. Taken

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together, our results suggested that the intrinsic apoptotic pathway would be activated by SpBOK

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to inhibit WSSV infection in mud crab.

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Acknowledgments

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The work was supported by grants from National Natural Science Foundation of China (41876152,

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31850410487, 31802341), Guangdong provincial project of Science and Technology

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(2017B020204003), the ‘Sail Plan’ Program for Outstanding Talents of Guangdong Province

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(14600605), support from Department of Education of Guangdong Province (2017KCXTD014)

291

and Guangdong Provincial Special Fund for Modern Agriculture Industry Technology Innovation

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Teams (2019KJ141).

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Table 1 The primers used in this study Primer name

Sequence (5`- 3`)

Objectives

BOK-F

CCCTTTCGTCCCTTTGTC

cDNA cloning

BOK-R

CTGCAAACACTGTCGCTACTC

ExBOK-F

GAAGTTCTGTTCCAGGGGCCCCTGGGATCCAT

Recombinant expression

GGCGAGCTTGCAACTG

ExBOK-R

GAAGTTCTGTTCCAGGGGCCCCTGGGATCCTC TTCCATGCCCATAGTC

Recombinant expression

PGEX-F

CCATCCTCCAAAATCGGATC

PGEX-R

GCCGCATCGTGACTGACTG

ß-ActinF

CAGCCTTCCTTCCTGGGTATGG

ß-ActinR

GAGGGAGCGAGGGCAGTGATT

RT-BOK-F

GGCGGGCCTCCTACATAAAAAACTG

RT-BOK-R

CTGTAGAGCTTCGGGTGGGTCC

WSSV-F

TATTGTCTCTCCTGACGTAC

WSSV-R

CACATTCTTCACGAGTCTAC

TaqMan probe

FAM-TGCTGCCGTCTCCAA-TAMRA

qRT-PCR

BOK-Oligo-1

GATCACTAATACGACTCACTATAGGGGGGAA

RNAi

qRT-PCR

qRT-PCR

qRT-PCR

GTATGCGAGGTGTTTT

BOK-Oligo-2

AAAACACCTCGCATACTTCCCCCCTATAGTGA GTCGTATTAGTGATC

BOK-Oligo-3

AAGGGAAGTATGCGAGGTGTTCCCTATAGTG AGTCGTATTAGTGATC

BOK-Oligo-4

GATCACTAATACGACTCACTATAGGGAACAC CTCGCATACTTCCCTT

GFP-Oligo-1

GATCACTAATACGACTCACTATAGGGGGCTA CGTCCAGGAGCGCACCTT

GFP-Oligo-2

AAGGTGCGCTCCTGGACGTAGCCCCCTATAGT GAGTCGTATTAGTGATC

GFP-Oligo-3

AAGGCTACGTCCAGGAGCGCACCCCCTATAG TGAGTCGTATTAGTGATC

GFP-Oligo-4

GATCACTAATACGACTCACTATAGGGGGTGC GCTCCTGGACGTAGCCTT

404

RNAi

405

Figure legends

406

Fig 1. The domain architecture of SpBOK. (A). Multiple alignments of SpBOK. Sc: Scylla

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paramamosain (MK779314), Pc: Palaemon carinicauda (AVC04857), Mc: Macrobrachium

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rosenbergii (CDI59404), Pv: Penaeus vannamei (ROT77702), Cr: Caligus rogercresseyi

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(ACO11568), Nl: Nilaparvata lugens (XP_022184369), Ea: Eurytemora affinis (XP_023338781),

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Pt: Parasteatoda tepidariorum (XP_015929024), Fo: Frankliniella occidentalis (XP_026286881),

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Acc: Apis cerana cerana (PBC33241); (B). Phylogenetic analysis (neighbor-joining analysis) of

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SpBOK; (C). Domain function prediction of SpBOK; (D). The 3D structure of SpBOK was

413

analyzed using the online SWISS-MODLE.

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Fig 2. SpBOK responses to the WSSV infection. (A). The tissue distribution of SpBOK was

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analyzed by qRT-PCR, and the relative expression of SpBOK in various tissues was compared

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with that in the hemocytes. (B). Expression profiles of SpBOK in hemocytes of crabs after the

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WSSV challenge. The crabs were injected with WSSV, the results are based on three parallel

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experiments and PBS served as a control. The efficiency of SpBOK was analyzed by qRT-PCR;

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(C-D). the relative WSSV copies in crabs challenged with WSSV at 0, 24, 48 and 72 hpi were

420

analyzed by qRT-PCR and western blotting, respectively. The results are based on three parallel

421

experiments and showed as mean values ± SD (*p<0.05, **p<0.01);

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Fig 3. Apoptosis of hemocytes induced by WSSV infection. (A). Detection of crab hemocyte

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apoptosis with Annexin V/PI. The crabs were challenged with either WSSV or PBS was used as

424

controls. The apoptotic rate of hemocytes was analyzed by flow cytometry; (B). The hemocyte

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apoptotic rate in Annexin-V/PI assay was shown in bar graphs. The results are based on three

426

parallel data and showed as mean values ± SD (*p<0.05, **p<0.01).

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Fig 4. SpBOK plays a significant role in Scylla paramamosain antiviral immunity. (A).

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SpBOK knockdown in hemocytes suppressed the expression of SpBOK. Crabs were injected with

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50 µg of siRNA, and the mRNA expression of SpBOK was analyzed at 24 hpi by qRT-PCR. siGFP

430

was used as a control. The results are based on three parallel data and shown as mean values ± SD

431

(*p<0.05, **p<0.01). (B). RNAi efficiency against SpBOK was determined by western blotting;

432

(C). The relative WSSV copies in siBOK crabs challenged with WSSV at 48 hpi were analyzed by

433

qRT-PCR. The groups of PBS, WSSV or siGFP were used as a control. The results are based on

434

three parallel data and expressed as mean values ± SD (*p<0.05, **p<0.01 compared with

435

controls at each time point).

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Fig 5. SpBOK regulates changes in mitochondrial membrane potential. (A). The siBOK crabs

437

were treated with WSSV, and hemocytes were collected at 48 hpi and differences in mitochondrial

438

membrane potential were measured by staining with JC-1. The control group was injected with

439

WSSV, PBS or co-injected with siGFP and WSSV. (B). ⊿ψm was measured by stained with JC-1,

440

and the fluorescent 530 and 590 nm, respectively, were detected. The results are based on three

441

parallel experiments and shown as mean values ± SD (*p<0.05, **p<0.01).

442

Fig 6. SpBOK mediates the activation of apoptosis. (A). The siBOK-injected crabs were

443

challenged with WSSV for 48 h; the hemocytes were stained with Annexin V/PI and used for

444

estimating the apoptotic rate using flow cytometry. PBS, WSSV single injection or co-treated with

445

siGFP and WSSV were the controls. (B). The hemocyte apoptotic rate of all treatments was

446

expressed in bar graphs. (C). Caspase-3 activity of the siBOK challenged with WSSV and the

447

control groups were detected. The results are based on three parallel data and shown as mean

448

values ± SD (*p<0.05, **p<0.01).

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Fig 7. The proposed model for the function of SpBOK in regulating WSSV infection and

450

apoptosis in mud crab.

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Fig 1 (A)

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(B)

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(C)

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(D)

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Fig 2

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Fig 3

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(A)

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(B)

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Fig 4 (A)

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(B)

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(C)

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Fig 5

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(A)

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(B)

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Fig 6

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(A)

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(B)

(C)

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Fig 7

Dear editor, The highlights are listed as following: SpBOK was significantly up-regulated after WSSV infection in vivo in mud crab. SpBOK activates the caspase signaling by reducing the mitochondrial membrane potential (⊿ψm). SpBOK inhibits WSSV infection by promoting intrinsic apoptosis in mud crabs.

With my best wishes! Shengkang Li