Dual oxidases participate in the regulation of hemolymph microbiota homeostasis in mud crab Scylla paramamosain

Accepted Manuscript Dual oxidases participate in the regulation of hemolymph microbiota homeostasis in mud crab Scylla paramamosain Zaiqiao Sun, Shufeng Hao, Yi Gong, Ming Zhang, Jude Juventus Aweya, Ngoc Tuan Tran, Yueling Zhang, Hongyu Ma, Shengkang Li PII:

S0145-305X(18)30305-7

DOI:

10.1016/j.dci.2018.08.009

Reference:

DCI 3237

To appear in:

Developmental and Comparative Immunology

Received Date: 11 June 2018 Revised Date:

9 August 2018

Accepted Date: 10 August 2018

Please cite this article as: Sun, Z., Hao, S., Gong, Y., Zhang, M., Aweya, J.J., Tran, N.T., Zhang, Y., Ma, H., Li, S., Dual oxidases participate in the regulation of hemolymph microbiota homeostasis in mud crab Scylla paramamosain, Developmental and Comparative Immunology (2018), doi: 10.1016/ j.dci.2018.08.009. 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

Dual oxidases participate in the regulation of hemolymph

2

microbiota homeostasis in mud crab Scylla paramamosain

3

Zaiqiao Sun1,2,a, Shufeng Hao1,2,a, Yi Gong1,2, Ming Zhang1,2, Jude Juventus Aweya1, 2, Ngoc Tuan

4

Tran1,2, Yueling Zhang1,2, Hongyu Ma1,2, Shengkang Li1,2*

5 6

1

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

7

2

8

a

10

*

Author for correspondence. E-mail: [email protected]

SC

These authors contributed equally to this paper

M AN U

9

Marine Biology Institute, Shantou University, Shantou 515063, China

RI PT

1

E-mail: [email protected]

11

Running title: DUOXs regulate mud crab hemolymph microbial homeostasis

13

Keywords: Scylla paramamosain; dual oxidases; hemolymph microbiota; homeostasis

EP AC C

14

TE D

12

1

ACCEPTED MANUSCRIPT ABSTRACT

16

Dual oxidases (DUOXs) were originally identified as NADPH oxidases (NOXs), found to be

17

associated with the reactive oxygen species (ROS) hydrogen peroxide (H2O2) production at the

18

plasma membrane and crucial in host biological processes. In this study, SpDUOX1 and

19

SpDUOX2 of mud crab (Scylla paramamosain) were identified and studied. Both SpDUOX1 and

20

SpDUOX2 are transmembrane proteins, including an N-signal peptide region and a peroxidase

21

homology domain in the extracellular region, transmembrane regions, and three EF

22

(calcium-binding region) domains, a FAD-binding domain, and a NAD binding domain in the

23

intracellular region. The SpDUOXs were expressed in all tissues examined, but mainly in

24

hepatopancreas, heart, and mid-intestine. The expression of the SpDUOXs in the hemolymph of

25

mud crabs was up-regulated after challenge with Vibrio parahemolyticus or LPS. RNA

26

interference (RNAi) of the SpDUOXs resulted in reduced ROS production in hemolymph. The

27

bacterial count increased in the hemolymph of mud crabs injected with SpDUOX1 or

28

SpDUOX2-RNAi, while the bacterial clearance ability of hemolymph significantly reduced. At the

29

phylum level, the phyla Bacteroidetes and Actinobacteria were significantly increased, while

30

Proteobacteria were significantly reduced following SpDUOX2 knockdown. There was a

31

significant increase in the relative abundance of the genera Marinomonas, Pseudoalteromonas,

32

Shewanella, and Hydrogenoph in SpDUOX2 depleted mud crabs compared with the controls. Our

33

current findings therefore indicated that SpDUOXs might play important roles in maintaining the

34

homeostasis in the hemolymph microbiota of mud crab.

SC

M AN U

TE D

EP

AC C

35

RI PT

15

2

ACCEPTED MANUSCRIPT 36

1. Introduction Aquatic invertebrates harbor a stable amount of bacteria in their hemolymph, which is

38

relatively lower than that in the intestinal tract, and with these bacteria potentially pathogenic to the

39

host under stressful (culture) conditions (Fagutao et al., 2012; Bruno et al., 1998). The hemolymph

40

also contains a number of molecules including antimicrobial peptides (AMPs) (i.e.

41

anti-lipopolysaccharide factors, crustins, penaeidins, and lysozymes), pattern recognition receptors

42

(PRRs) (i.e. lectin), oxidase enzymes (i.e. prophenoloxidase). Among all these molecules

43

(Fredrick et al., 2012; Rosa et al., 2010), AMPs are known to be important in inhibiting

44

hemolymph microbiota proliferation and therefore maintains homeostasis in crustaceans (Wang et

45

al., 2013; Wang et al., 2014). It has been reported that after RNA interference (RNAi)-based gene

46

silence through dsRNA injection, the bacterial counts in the hemolymph of kuruma shrimp

47

(Marsupenaeus japonicus) was significantly increased, leading to high mortality (Wang et al.,

48

2014). It has also been shown that the growth of bacteria increased in the hemolymph of M.

49

japonicus after RNAi of lysozyme (dsLYZ injection) or prophenoloxidase (dsproPO injection)

50

(Kaizu et al., 2011; Fagutao et al., 2009; Wang et al., 2015). Furthermore, it has been shown that

51

the immune deficiency pathway and reactive oxygen species (ROS) play important roles in the

52

immune system, by maintaining gut microbiota homeostasis in Drosophila (Ha et al., 2005). ROS

53

acts as immune effectors, exerting microbicidal activity in gut immunity (Ha et al., 2005),

54

although ROS induced by oxidative enzymatic reactions was previously considered to be harmful

55

to organisms. However, when the phagocyte oxidase (gp91phox/NOX2) was discovered as the

56

enzyme responsible for the “respiratory burst” (Salathe et al., 1997), ROS was implicated in

57

specialized cellular functions in various subcellular compartments (Kawahara et al., 2007). ROS is

58

produced by membrane NADPH oxidase (NOX) enzymes, which comprise of seven NOX family

59

members (NOX1-NOX5, DUOX1 and DUOX2) in the human genome (Ryu et al.,2010),

60

characterized by a conserved catalytic core responsible for transmembrane electron transfer from

61

intracellular electron donors to the extracellular compartment to produce superoxide (O2-) or H2O2

62

(De et al., 2014). Dual oxidases (DUOXs) are NOX family members, originally identified as

63

thyroid oxidases and later renamed as “Dual oxidase” based on the presence of peroxidase-like

64

and NADPH oxidase domains on the N-terminals and C-terminals, respectively (De et al., 2015;

AC C

EP

TE D

M AN U

SC

RI PT

37

3

ACCEPTED MANUSCRIPT Xie et al., 2014; Ohye et al., 2010; Dupuy et al., 1999; Edens et al., 2001). DUOXs have been

66

found in many tissues, including cochlea, phagocytes, respiratory tract, and intestinal tract of

67

different animals (Guichard et al., 2006). In mice, the expression of DUOX2 is contingent upon

68

induction of different signaling pathways in the ileum and colon epithelium during gut microbiota

69

invasion (Sommer et al., 2015). DUOXs are associated with ROS hydrogen peroxide (H2O2)

70

production at the plasma membrane, which is crucial in biological processes including

71

thyroid-hormone biosynthesis, tyrosine-crosslinking in the cuticle and fertilization envelope, and

72

immunity (Sasakura et al., 2017). In mammals, DUOXs have a peroxidase-like domain, but have

73

no peroxidase activity because they lack the histidine that is essential for peroxidase activity

74

(Edens et al., 2001; Furtmüller et al., 2006; Sumimoto et al., 2005). However, in Drosophila, the

75

extracellular peroxidase homology domain (PHD) has an enzyme activity that converts H2O2 to

76

HOCl in the presence of chlorideion (Ha et al., 2005). To date, few studies have been reported on

77

the functions of DUOXs in invertebrates. In Drosophila, a specific RNAi sequence has been found

78

to be involved in an increased mortality rate (Ha et al., 2005; Leto et al., 2009), while in the

79

midgut of mosquito (Anopheles gambiae), DUOX decreased permeability to immune elicitors and

80

protected the host against bacteria and Plasmodium parasites (Kumar et al., 2010). In kuruma

81

shrimp (M. japonicas), knockdown of MjDUOXs decreased the ROS level and increased the

82

bacterial number in the intestine (Yang et al., 2016).

TE D

M AN U

SC

RI PT

65

Mud crab (S. paramamosain) is a commercially important crustacean species cultured in the

84

southeastern coast of China (Li et al., 2008). Recently, frequent disease outbreaks in culture

85

organisms could affect the sustainable development of the aquaculture industry (including mud

86

crab aquaculture). Thus, a better understanding of the immune system (especially the microbial

87

homeostasis in the hemolymph) of mud crab is needed. So far, the functions of DUOXs in the

88

immune system in mud crab have not been well understood. In this study, SpDUOXs (including

89

DUOX1and DUOX2) were cloned and characterized in different tissues of healthy crab and upon

90

immune challenges. The results revealed that SpDUOXs decreased the ROS level and increased

91

the bacterial counts in the hemolymph of mud crab. The bacterial clearance ability of the

92

hemolymph in mud crab was significantly reduced after SpDUOXs RNAi. The results suggested

93

that SpDUOXs played an important role in maintaining the homeostasis of the bacterial population

94

in the hemolymph of mud crab.

AC C

EP

83

4

ACCEPTED MANUSCRIPT 95

2. Materials and methods

96

2.1 Challenge test and sampling A total of 48 healthy mud crabs (approximately 100 g each) were purchased from a crab farm

98

in Niutianyang (Shantou, Guangdong, China), and acclimatized to laboratory conditions (seawater

99

with a salinity of 8‰ and temperature of 25oC) for one week. After acclimatization, the crabs were

100

divided into experimental and control groups and used for a challenge test. For the experimental

101

groups, crabs were injected with 200 µL Vibrio parahemolyticus (1×107 colony-forming unit (cfu

102

mL-1) or 200 µL lipopolysaccharide (LPS, Sigma, USA) (0.5 mg mL-1) at the base of the fourth leg

103

of each crab. In the control, crabs were injected with the same volume of sterile

104

phosphate-buffered saline (PBS) solution (1L; 0.14 M NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and

105

1.8 mM KH2PO4. The experiments were conducted under laboratory conditions, and hemolymph

106

collected from crabs using a disposable needle and syringe (1 mL) at 0, 6, 12, 24, 48, and 96 hour

107

post injection (hpi), into tubes containing ice-cold acid citrate dextrose (ACD) anticoagulant

108

buffer (1.32% sodium citrate, 0.48% citric acid, and 1.47% glucose). Samples were centrifuged at

109

800 ×g for 20 min at 4oC to separate the hemocytes, which was then used for RNA extraction

110

using a test kit (TRIzol® Reagent, Ambion, USA). Other tissues including stomach, muscle,

111

subcuticular epidermis, gills, hepatopancreas, intestines, and heart were then quickly collected,

112

rinsed with 0.1% diethylpyrocarbonate (DEPC)-treated water and immediately dipped into liquid

113

nitrogen. The tissues were sampled from at least three mud crabs per group for subsequent total

114

RNA extraction.

115

2.2. RNA extraction and first-strand cDNA synthesis

SC

M AN U

TE D

EP

AC C

116

RI PT

97

Total RNA was extracted from the collected tissues (except hemocytes) using the Total RNA

117

Rapid Extraction Kit (Feijie, Shanghai, China) following the manufacturer’s protocol. The purity

118

and quality of the RNA samples was ascertained using 1.0% (w/v) agarose-gel electrophoresis

119

while the concentration was measured with a Nanodrop® ND-1000 spectrophotometer (LabTech,

120

Holliston, MA) at absorbance 260 nm/280 nm (A260/280). A total of 5 mg RNA from the

121

hemocytes was reversely transcribed with the M-MLV First-Strand cDNA Synthesis Kit

122

(Invitrogen, USA) for subsequent full-length SpDUOX cloning. Total RNA extracted from other

123

tissues was used as templates to synthesize the first-strand cDNA using PrimerScript RT Reagent 5

ACCEPTED MANUSCRIPT 124

Kit with gDNA Eraser (Takara, Dalian, China), for quantitative real-time PCR (qRT-PCR).

125

2.3 Cloning the full-length cDNA of SpDUOX1 and SpDUOX2 The partial cDNA sequence of SpDUOX1 and SpDUOX2 of mud crab was obtained from

127

our previous high-throughput transcriptome data. The complete SpDUOXs cDNA was amplified

128

through 3’RACE and 5’RACE PCR with the SMARTerTM RACE cDNA Amplification Kit

129

(Clontech, USA), using touch-down PCR and nested PCR strategy with specific primers (Table 1).

130

For the RACE PCR, 28 µL of sterile distilled water, 8 µL of dNTP mixture, 5 µL of 10×LA PCR

131

Buffer II (Mg2+ Plus), and 0.5 µL of LA Taq (TaKaRa, Dalian, China) were added to a total

132

reaction volume of 50 µL. Similarly, the total volume of touch-down PCR for 3’RACE or

133

5’RACE was 50 µL containing 41.5 µL of RACE Master Mix, 1 µL of DUOX-GSP3-1 or

134

DUOX-GSP5-1 primer, 5 µL of UPM (0.4 µM Long Universal Primer and 2 µM Short Universal

135

Primer), and 2.5 µL of cDNA template. The touch-down PCR was programmed as follows: 5

136

cycles at 94oC for 30 s, 72 oC for 2min 30s; 5 cycles at 94 oC for 30 s, 68oC (3’RACE) or 70oC

137

(5’RACE) for 30 s, 72oC for 2min 30 s; and 38 cycles (3’RACE) or 27 cycles (5’RACE) of 94oC

138

for 30 s, 64oC (3’RACE) or 68oC (5’RACE) for 30 s, 72oC for 2.5 min. The product of the

139

touch-down PCR was used as the template for the subsequent nested PCR. The nested PCR was

140

also performed in a total reaction volume of 50 µL including 47.5 µL of RACE Master Mix, 1 µL

141

of DUOX-GSP3-2 or DUOX-GSP5-2 primer, 1 µL of NUP primer, and 0.5 µL of templates. The

142

nested PCR condition was of 94oC for 3 min followed by 38 cycles (3’RACE) or 30 cycles

143

(5’RACE) at 94oC for 30 s, 64oC (3’RACE) or 68oC (5’RACE) for 30s, 72oC for 2.5 min; and a

144

final extension at 72oC for 10 min. Purified DNA fragment was cloned into the pMD®19-T vector

145

(TaKaRa, Dalian, China) and then transformed into Escherichia coli. Positive recombinant clones

146

were identified by PCR screening with M13R and M13F primers and sequenced by a commercial

147

company (BGI, Shenzhen, China).

148

2.4 Bioinformatics analysis

AC C

EP

TE D

M AN U

SC

RI PT

126

149

Sequence (nucleotide and amino acid) homology analysis was carried out with the online

150

BLAST program (http://www.ncbi.nlm.nih.gov/blast/). The deduced amino acid sequence was

151

obtained with the ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/orfig.cgi). Multiple protein

152

sequence alignment was performed with Clustal W2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/) 6

ACCEPTED MANUSCRIPT and analyzed on DNAMAN. MEGA 5.10 was used to construct phylogenetic neighbor-joining

154

(NJ) trees. The web-based SMART program (http://smart.embl-heidelberg.de/) was used to

155

detect the protein domains in the SpDUOXs. The three-dimensional (3D) structure of SpDUOXs

156

was predicted by I-Tasser server (http://zhanglab.ccmb.med.umich.edu/I-TASSER/).

157

2.5 Quantitative RT- PCR assay

RI PT

153

The quantitative RT- PCR (qRT-PCR) analysis was carried out using the SYBR®Premix Ex

159

Taq™ II Kit (Takara, Dalian, China) on a LightCycler® 480 (Roche, USA). First-strand cDNA

160

was first synthesized as described above and used as templates for qRT-PCR with specific primers

161

(Table 1). β-actin was used as an internal control. A total reaction volume of 20 µL containing 10

162

µL of SYBR® Premix Ex Taq™ II, 2 µL of the four-fold diluted cDNA, 0.8 µL (10 µM) each of

163

forward and reverse primer, and 6.4 µL of ultra-pure water. The amplification procedure included

164

a denaturation step of 95oC for 30 s, and 40 cycles of 95oC for 5 s, 60oC for 20 s, followed by a

165

melting curve analysis from 65oC to 95oC. Triplicate samples were used in each experiment. Data

166

were analyzed using the LightCycler 480 software (Roche, USA). Relative expression of

167

SpDUOXs was determined using the 2-∆∆Ct algorithm.

168

2.6 RNA interference

TE D

M AN U

SC

158

Small interfering RNAs (siRNAs) were used to knockdown SpDUOXs. The siRNAs

170

(siDUOX1, siDUOX2, and siGFP) were chemically synthesized using an in vitro Transcription T7

171

Kit (TaKaRa, Dalian, China) with the primers listed in Table 1. The final concentration of siRNA

172

was diluted with DEPC-water to 125 µg mL-1. To determine efficiency of the RNA interference

173

(RNAi), 65 healthy mud crabs (approximately 25-30g each) were acclimatized to laboratory

174

conditions (as above described) before further processing. Crabs were divided into two groups

175

(with 20 individuals per group), and injected at the base of the third leg with 200 µL siGFP (25 µg

176

crab-1) negative control or with 200 µL of either siDUOX1 or siDUOX2 (25 mg crab-1). At 24, 36,

177

and 48 h after the siRNA injection, hemocytes (of three mud crabs per group) were collected to

178

determine the expression of SpDUOX1 and SpDUOX2 by qRT-PCR using the primers

179

Q-DUOX1R, Q-DUOX1F, Q-DUOX2R, and Q-DUOX2F (Table 1). β-actin was used as a

180

reference gene.

181

2.7 Bacterial count and ROS assay in hemolymph after SpDUOXs knockdown

AC C

EP

169

7

ACCEPTED MANUSCRIPT The crabs were randomly divided into four groups, for the experimental groups, siDUOX1,

183

siDUOX2 or mixture of siDUOX1 and siDUOX2 were injected into mud crabs. The crabs were

184

injected with siGFP were used as the controls. The hemolymph from four mud crabs per group

185

was collected at 24 h, 36 h, 48h after siRNA injection. Hemocytes were filtered from the

186

hemolymph first with a 5-µm mesh membrane (TMTP02500, Millipore), and then with a 0.2-µm

187

mesh membrane (GTBP02500, Millipore). The 0.2-µm mesh membrane was then stained with

188

SYBR® Green I solution (1:40 v/v SYBR® Green I in 1×Tris-EDTA buffer) for 20 min. The stain

189

solution was removed; membrane placed onto a glass slide, and 30 µL of glycerine (10% v/v)

190

added as an antifade agent. Cells were counted at 1000x magnification using a fluorescence

191

microscope (Zeiss, Axioplan 2 Imaging, Germany) with a blue filter set (Zhang et al., 2018). The

192

ROS level of hemolymph was detected using the Hydrogen Peroxide Assay Kit (Nanjing

193

Jiancheng, Jiangsu, China) (Zhang et al., 2015).

194

2.8 Bacterial clearance assay after SpDUOXs knockdown

M AN U

SC

RI PT

182

Laboratory acclimatized mud crabs were randomly selected and divided into four groups, i.e.,

196

siDUOX1, siDUOX2, siGFP, and DEPC-treated H2O groups. Crabs were then injected with 200

197

uL crab-1 of the respective siRNAs or DEPC-treated H2O at the abdominal segment. Twenty-four

198

hours after siRNA injection, 200 mL of V. parahemolyticus (1×106 cfu mL-1) was injected into

199

each crab. Hemolymph was collected 6 h after the V. parahemolyticus injection, diluted with

200

0.85% NaCl, and cultured on 2216E solid plates (containing 0.5% tryptone, 0.1% yeast extract,

201

1.5% agar, 0.01% FePO4, and seawater) at 37 oC for 36 h. The number of colonies were counted

202

and recorded.

203

2.9 Sequencing of hemolymph microbiota after SpDUOX2 knockdown

EP

AC C

204

TE D

195

Twenty-four hours after SpDUOX2 RNAi, hemolymph was taken from mud crabs injected

205

with siDUOX2 or siGFP. The hemolymph microbial DNA was extracted using a E.Z.N.A DNA

206

/RNA Isolation Kit (OMEGA, USA) according to the manufacturer’s protocol. Quality and

207

quantity of the extracted DNA were evaluated by agarose gel electrophoresis and NanoDrop 1000

208

spectrophotometer [38]. The 16S rRNA gene was amplified by PCR using universal primers (27F

209

and 1492R) (Table 1). The PCR reactions were performed in triplicate in a 20 µL mixture

210

containing 10 µL of 2× PCR mix (DSBIO), 1 µL of each primer (10 µM), 2 µL of DNA templates, 8

ACCEPTED MANUSCRIPT and 6 µL of sterile double-distilled water. The PCR cycling conditions were as follows: 95 °C for

212

5 min, followed by 30 cycles at 95°C for 30 s, 55°C for 30 s, and 72 °C for 90 s and a final

213

extension at 72 °C for 10 min. Quality of the PCR products were electrophoresed on 1% agarose

214

gels. The samples were analyzed by sequencing the V3-V4 regions (about 465 bp) of 16S rRNA

215

genes on an Illumina HiSeq 2500 platform (2×250 bp; Illumina, USA). Data analysis was

216

performed as described elsewhere (Zhang et al., 2018). Three samples from siDUOX or siGFP

217

were removed due to low quality of the sequencing data.

218

2.10 Statistical analysis

RI PT

211

Data represents the results of at least three independent experiments. All data are expressed

220

as means ±S.D. The data was subjected to one-way ANOVA analysis using Origin Pro 8.5

221

followed by t-test, with P<0.05 considered statistically significant and extremely significant at

222

P<0.01.

M AN U

SC

219

AC C

EP

TE D

223

9

ACCEPTED MANUSCRIPT 224

3. Results

225

3.1. Characterization of SpDUOX1 and SpDUOX2 cDNA The complete cDNA sequence of SpDUOX1 consists of 4786 bp in length, containing a

227

5’-untranslated region (5’-UTR) of 38 bp, an open reading frame (ORF) of 4494 bp encoding

228

1497 deduced amino acids (aa), and a 254 bp 3’-UTR (Fig.S1 A). The cDNA sequence of

229

SpDUOX1 has been deposited at NCBI GenBank under the accession number MH023417. The

230

estimated molecular mass of SpDUOX1 is 173 kDa, and the theoretical pI is 8.50. SpDUOX1 is a

231

transmembrane protein, with an N-signal peptide region (20 aa), a peroxidase homology domain

232

(PHD, 526 aa) in the extracellular region, six transmembrane regions, three EF (calcium-binding

233

region) domains (110 aa), a FAD binding domain (104 aa) and a NAD binding domain (157 aa) in

234

the intracellular region (Fig. S2). On the other hand, the complete cDNA sequence of SpDUOX2

235

is 5093 bp in length, containing a 5’-UTR of 46 bp, an ORF of 4713 bp encoding 1570 aa, and a

236

335 bp 3’-UTR with a predicted alternative polyadenylation signal site (AATAAA) and a poly(A)

237

tail (Fig.S1 B). The sequence of SpDUOX2 is deposited at NCBI GenBank under the accession

238

number MG840835. SpDUOX2 is also a transmembrane protein, with an N-signal peptide region

239

(23 aa) and transmembrane region with a peroxidase homology domain (PHD, 552aa) in the

240

extracellular region, seven transmembrane regions, three EF (calcium-binding region) domains

241

(110 aa), a FAD binding domain (104 aa), and a NAD binding domain (155 aa) in the intracellular

242

region (Fig. S2).

243

3.2. Comparison of domain architectures and 3D structures of DUOXs

EP

TE D

M AN U

SC

RI PT

226

The protein domains of DUOXs from different species were compared (Fig. S2). SpDUOX1

245

and MjDUOX1 have 6-transmembrane regions, However, SpDUOX2 and MjDUOX2 have 7-

246

transmembrane regions. SpDUOXs and MjDUOXs both have 3 EF hand regions. The 3D

247

structures were also predicted using an online software. The overall structures of SpDUOX1 and

248

SpDUOX2 are different from each other (Fig. S3 A and B). The difference illustrates that

249

SpDUOX1 and SpDUOX2 might be involving in different physiological processes. However, the

250

PHD domain responsible for ROS production shows high similarity (Fig. 3S C and D). This

251

indicates that SpDUOX1 and SpDUOX2 both can generate ROS.

AC C

244

252 10

ACCEPTED MANUSCRIPT 253

3.3. Sequence comparison and phylogenetic tree analysis Phylogenetic tree analysis using MEGA 5.0 software (Fig. 1) showed that all DUOXs fell

255

into three clusters with cluster 1 made up of vertebrates DUOXs, cluster 2 comprising of

256

SpDUOX1 and a number of invertebrates’ DUOXs (i.e., insects, Caenorhabditis elegans, and M.

257

japonicas), and cluster 3 consisting of SpDUOX2 and other invertebrates’ DUOXs. SpDUOX1

258

had the highest homology of 86% with MjDUOX1 (GenBankr: BAM76968) of M. japonicus,

259

followed by 68.2 % with CfDUOX (EFN74201) from Camponotus floridanus, and 40.5% with

260

CeDUOX (AAF71303) of C. elegans. On the other hand, SpDUOX2 was almost similar to

261

MjDUOX2 (ANA91274) of M. japonicas with an identity of 55.7%, CfDUOX (EFN70161) of C.

262

floridanus with 37 %, and CgDUOX (EKC42615) of Crassostrea gigas with 28.2 % identity. The

263

homology between SpDUOX1 and SpDUOX2 was only 28%. Multiple sequence alignment

264

showed a higher similarity between SpDUOXs and MjDUOXs (ranging from 55.7 % to 86.2 %)

265

(Fig. S4).

266

3.4. Tissue distribution of SpDUOXs transcripts

M AN U

SC

RI PT

254

To determine the tissue distribution of SpDUOXs in mud crab, qRT-PCR was employed to

268

analyze its transcriptional levels in various tissues including hemocytes, hepatopancreas, muscle,

269

stomach, mid-intestine, subcuticular epidermis, gill, and heart. The results showed that SpDUOX1

270

and SpDUOX2 were expressed in all examined tissues, but mainly in hepatopancreas, heart, and

271

mid-intestine. However, low expression of SpDUOX1 (Fig. 2A) was observed in hemocytes and

272

muscle, while gill and muscle expressed low levels of SpDUOX2 (Fig. 2B).

273

3.5. Expression of SpDUOXs transcripts after immune challenges

EP

AC C

274

TE D

267

The expression profile of SpDUOXs in hemocytes and the hepatopancreas of mud crabs

275

after challenge with V. parahemolyticus or LPS at different time points was investigated using

276

qRT-PCR. The results revealed that SpDUOX1 was significantly (P<0.05) up-regulated in

277

hemocytes and in hepatopancreas at 6 and 12 hpi with V. parahemolyticus or at 6 hpi with LPS,

278

while there was no significant difference (P>0.05) between the experimental groups (V.

279

parahemolyticus or LPS injected) and the controls at the other time points (Fig. 2 C, E). The

280

expression of SpDUOX1 in hemocytes was similar to that in hemocytes post challenge with V.

281

parahemolyticus or LPS (Fig. 2 D). On the other hand, SpDUOX2 was significantly (P<0.05) 11

ACCEPTED MANUSCRIPT up-regulated in both hemocytes and the hepatopancreas at 6 hpi, significantly (P<0.05)

283

down-regulated in hepatopancreas at 12 hpi with V. parahemolyticus and LPS. The H2O2 level was

284

also detected after V. parahemolyticus infection in the hemolymph, the H2O2 level obviously

285

increased at 6 hpi (Fig. 2G). These results suggest that both SpDUOXs are responsible for ROS

286

production and play an important role in innate immunity.

287

3.6. Effects of SpDUOXs silencing on bacterial count and ROS production

RI PT

282

The efficiency of the DUOX siRNAs to silence SpDUOXs was evaluated at the mRNA

289

transcript level using qRT-PCR. At 24 h post siRNA injection, the qRT-PCR results revealed a

290

significant decrease in the expression of SpDUOX1 (78%) and SpDUOX2 (67.1%) in the

291

hemocytes of mud crabs compared with the controls (Figs. 3A and 3B). We confirmed that the

292

siSpDUOX1 treatment did not have off-target effects to siSpDUOX2, and siSpDUOX2

293

knockdown also does not affect the expression of SpDUOX1 (Figs. S5). Meanwhile, effects of

294

SpDUOXs silencing on bacterial count and ROS production were detected. The production of

295

H2O2 in crab hemocytes was also significantly decreased at 24 h post siSpDUOXs injection (Fig.

296

3C), which indicated that SpDUOXs were responsible for ROS produce. On the other hand, a

297

significant (P<0.05) increase in bacterial count was observed at 24 h post siSpDUOXs injection,

298

but recovered to baseline level at 48 h (Fig. 3D). The double knockdown experiments were

299

performed and the results showed that the hemolymph bacterial count in double knockdown

300

individuals (SiSpDUOX1+ X2) had no significant change compared with that in SpDUOX2

301

knockdown groups at 24h and 36h after siRNA injections (Fig. 3D).

302

3.7. Silencing of SpDUOX1 or SpDUOX2 reduces bacterial clearance in mud crab

M AN U

TE D

EP

AC C

303

SC

288

After RNAi of SpDUOX1 or SpDUOX2, the mud crab were infected with V.

304

parahaemolyticus to analyze gene function in crab hemolymph, bacteria numbers were counted in

305

a number of mud crabs. It was observed that, there was a higher total bacterial count in

306

siSpDUOX1 and siSpDUOX2 treated crabs as compared to siGFP treated crabs. When infected

307

with V. parahaemolyticus, the total bacterial number in siSpDUOX1 and siSpDUOX2 treated

308

crabs increased significantly compared to the control groups, for siSpDUOX2 treated crabs, the

309

total bacterial number increased dramatically when infected with V. parahaemolyticus as

310

compared to the negative control groups (Fig. 4). 12

ACCEPTED MANUSCRIPT 311

3.8. Hemolymph microbial community composition in mud crabs depleted of SpDUOX2 The microbial community compositions in the hemolymph of mud crabs silenced of

313

SpDUOX2 were investigated. The results generated a total of 737737 quality-filtered tags,

314

including 13400 OTUs, from 10 specimens (Table 2). The NMDS plot and PCoA plot showed a

315

clear separation of microbial communities between the siGFP and siSpDUOX2 groups, with

316

significant differences between the two groups in terms of shannon index and the microbial

317

community structure, which were divided into two clusters (Fig. 5A-C). The high throughput

318

sequencing results showed that Proteobacteria. Bacteroidetes and Firmicutes in the hemolymph

319

were obviously changed after RNAi of SpDUOX2，while Bacteroidetes and Actinobacteria were

320

in crab hemolymph higher relative abundance in SpDUOX2-RNAi groups than those in the

321

control groups，with an increase of 9.98%-12.8% and 11.4%-18.3%, respectively. Meanwhile,

322

Proteobacteria has lower relative abundance than the control, with a decrease of 65.01%-49.9%

323

(Figure 5D). Relative abundance of the major genera Flavobacterium, Arcobacter, Lactobacillus,

324

Pseudomonas,

325

Hydrogenophaga, Acinetobacter, Marinomonas, and Shewanella in the siDUOX2-injected group

326

and the siGFP-injected group (Figure 5E). And SpDUOX-RNAi groups had a higher relative

327

abundance of Flavobacterium and Arcobacter than the controls. There was a significant increase

328

in relative abundance of the genera Marinomonas (1.37 % vs. 0.15 %), Pseudoalteromonas

329

(1.07 % vs. 0.31 %), Shewanella (0.66 % vs. 0.22 %), and Hydrogenoph (0.62 % vs. 0.13 %) in

330

siDUOX-injected group compared with that in siGFP-injected controls (P<0.05) (Table 3). Venn

331

diagram analysis showed that the number of detected unique microbiota harboring the hemolymph

332

of mud crabs injected with siDUOX was more than that of the siGFP-injected group (Figure 5F).

333

The data indicated that the crabs with reduced SpDUOX2 gene expression showed a profound

334

difference in bacterial community composition.

SC

M AN U

Vibrio,

Lactococcus,

Bacteroides,

Pseudoalteromonas,

EP

TE D

Acinetobacter,

AC C

335

RI PT

312

13

ACCEPTED MANUSCRIPT 336

4. Discussion In this study, we identified the dual oxidases (SpDUOX1 and SpDUOX2) in the mud crab

338

Scylla paramamosain, and examined their functions in the regulation of hemolymph microbiota

339

homeostasis in the host. Our results revealed that SpDUOXs were associated with ROS production

340

and played roles in microbial homeostasis in crab hemolymph. It has been reported that DUOXs

341

are distributed similarly in various tissues in mammals, insects, and crustacean. For instance, in

342

human, DUOX transcripts have been reported in brain, heart, kidney, pancreas, skin, liver, and

343

prostate (Ha et al., 2005; De et al., 2000). DUOX1 and DUOX2 are expressed in the epithelial

344

cells of the gastrointestinal and respiratory tracts, but not in thyroid tissue (Juhasz et al., 2009).

345

Recently, in the silkmoth, Bombyx mori, BmDUOX was shown to be expressed in many tissues,

346

with high expression in testis, ovary, fat body and integument (Oliveira et al., 2013). In the case of

347

aquatic animals, the MjDUOX1 and MjDUOX2 of kuruma shrimp (M. japonicus) were highly

348

expressed in gills, stomach, hepatopancreas, ovaries, hematopoietic organs, and intestine (Yang et

349

al., 2016; Inada et al., 2013). These findings support the findings of the present study where both

350

SpDUOX1 and SpDUOX2 were widely distributed in different tissues (i.e., hemocytes,

351

hepatopancreas, muscle, stomach, mid-intestine, subcuticular epidermis, gill, and heart) of mud

352

crabs. Given that our results showed the expression of SpDUOXs in hemocytes, suggest their

353

crucial importance in regulating microbiota homeostasis in the hemolymph of mud crab.

TE D

M AN U

SC

RI PT

337

In human blood circulation, ROS seems to be important in stimulating immune responses

355

during acute wound healing (Dunnill et al., 2017). The roles of ROS such as O2- and H2O2 in

356

innate immunity in excluding invasive microbes have also been reported in crustaceans (Muñoz et

357

al., 2000). Muñoz found the association between O2- production with E. coli quantity in the

358

hemocytes of Pacific white shrimp (L. vannamei) (Muñoz et al., 2000). Herein, to the best of our

359

knowledge, this is the first study on the role of DUOXs in the hemolymph of mud crab, although

360

the genes encoding ROS-producing enzymes in shrimp have been well studied (Inada et al., 2013).

361

Here, SpDUOXs were found to be up-regulated in both hemocytes and hepatopancreas of mud

362

crab at 6 or 12 hpi with either V. parahemolyticus or LPS, suggesting their involvement in the

363

immune response to bacterial infection, especially in the first stages of infection. These results are

364

generally consistent with previous findings in kuruma shrimp (M. japonicas) after challenge with

AC C

EP

354

14

ACCEPTED MANUSCRIPT either white spot syndrome virus (WSSV) or V. parahemolyticus (Yang et al., 2016; Inada et al.,

366

2013), or in mice with microbial dysbiosis in the intestinal epithelial mucosa (Grasberger et al.,

367

2015). Additionally, in the human respiratory epithelium, DUOX has been reported to be the main

368

isoform responsible for the production of extracellular H2O2 in response to LPS or flagella (Koff

369

et al., 2008; Linderholm et al., 2010).

RI PT

365

It was observed here that, SpDUOX1 and SpDUOX2 knockdown by RNAi resulted in less

371

ROS production, thus suggesting that they might play important roles in the hemolymph microbial

372

homeostasis of mud crabs. SpDUOXs induced the generation of ROS in hemocytes, suggesting

373

their importance in inhibiting pathogens invading the hemolymph of mud crab. Previous reports

374

have indicated that mammalian DUOX functions to generate sufficient H2O2 that supports the

375

production of bactericidal hypothiocyanite in the presence of airway surface liquid components

376

(Moskwa et al., 2007). Both hemocytes and hepatopancreas are important tissues in the immune

377

system of crustaceans (Cerenius et al., 2004). In this study, depletion of SpDUOXs increased the

378

hemolymph bacterial count of mud crabs, suggesting that hemolymph was an important tissue

379

responsible for immune response in mud crab (Zhang et al., 2015). This observation is in

380

agreement with one previous study, where an increased population of microbiota was observed in

381

the intestine of kuruma shrimp (M. japonicas) after MjDUOX RNAi (Yang et al., 2016). The

382

microbiota harboring the intestine of mud crabs after an RNAi has not yet been explored and

383

therefore merits further investigations.

EP

TE D

M AN U

SC

370

When the technique of high-throughput sequencing was applied to investigate the changes in

385

the hemolymph microbiota population of mud crabs after siDUOX RNAi, the results showed the

386

major hemolymph microbiota to include the genera Flavobacterium, Arcobacter, Lactobacillus,

387

Pseudomonas,

388

Hydrogenophaga, Acinetobacter, Marinomonas, and Shewanella, which is almost similar to the

389

results obtained previously (Zhang et al., 2018; Givens et al., 2013). Interestingly, many

390

predominant potential microbial pathogens in other aquatic invertebrates were found in the

391

hemolymph of mud crabs, including Vibrio, Acinetobacter, Aeromonas, Flavobacterium,

392

Arcobacter, and Tenacibaculum (Longeon et al., 2004; Williams et al., 2017; Carolin et al., 2014),

393

members of which have caused great economic losses in the aquaculture industry (Lafferty et al.,

394

2015). In this study, we found that the composition and richness of the hemolymph microbial

AC C

384

Acinetobacter,

Vibrio,

Lactococcus,

15

Bacteroides,

Pseudoalteromonas,

ACCEPTED MANUSCRIPT community changed markedly in SpDUOX-RNAi individauls. SpDUOX-RNAi crabs had a higher

396

relative abundance of Flavobacterium and Arcobacter than the controls. In the case of S.

397

paramamosain, some Acinetobacter spp are opportunistic pathogens as in human and some

398

aquaculture animals (Xiong et al., 2015), while some Aeromonas spp. and Flavobacterium spp,

399

are also frequently isolated from diseased fish and shrimps, causing haemorrhagic septicaemia, fin

400

rot or furunculosis (Fečkaninová et al., 2017; Anson et al., 2015). Similarly, Arcobacter and

401

Tenacibaculum are known pathogens that are reported to have caused considerable economic

402

losses in marine fish and mollusks aquaculture (Burioli et al., 2018; Collado et al., 2014). In any

403

case, the hemolymph of mud crabs also harbors potential probiotics such as Pseudoalteromonas,

404

which protect the host from infection (Desriac et al., 2014). A previous study reported that the

405

abundance of Pseudoalteromonas in the hemolymph of C. angulate was able to produce

406

antimicrobials beneficial to the host against Vibrio infection (Longeon et al., 2004). However,

407

rapid proliferation of these microorganisms in the hemolymph may lead to diseases, which has

408

been found previously in mud crab (S. paramamosain) (Zhang et al., 2018) and kuruma shrimp (M.

409

japonicas) (Kaizu et al., 2011; Fagutao et al., 2009; Wang et al., 2015). It was indicated that the

410

crabs with SpDUOX2 silence led to a profound change in bacterial community composition in our

411

study, and the change might be a reflection of difference of the resistance of microbes in

412

hemolymph to ROS killing activity in mud crab.

TE D

M AN U

SC

RI PT

395

In conclusion, SpDUOX1 and SpDUOX2 were identified in S. paramamosain, and were

414

shown to be involved in ROS production during hemolymph immune response. Depletion of

415

SpDUOX1 and SpDUOX2 increased hemolymph bacterial count of mud crabs, as the bacterial

416

clearance ability of hemolymph was significantly reduced. A significant increase in the relative

417

abundance of the genera Marinomonas, Pseudoalteromonas, Shewanella, and Hydrogenoph was

418

observed following siDUOX2 treatment. The findings here therefore indicated that SpDUOXs

419

may play important roles in maintaining the bacterial homeostasis in the hemolymph of mud

420

crabs.

AC C

EP

413

421

16

ACCEPTED MANUSCRIPT 422 423

Acknowledgments The work was supported by grants from National Natural Science Foundation of China

424

(41641053,

41376176),

425

(2017B020204003), the “Sail Plan” Program for Outstanding Talents of Guangdong Province

426

(14600605)

427

(2017KCXTD014).

support

from

provincial

Department

project

of

of

Education

Science

of

and

Technology

Guangdong

Province

RI PT

and

Guangdong

AC C

EP

TE D

M AN U

SC

428

17

ACCEPTED MANUSCRIPT 429

References

430

Fagutao, F.F., Maningas, M.B., Kondo, H., Aoki, T., Hirono, I., 2012. Transglutaminaseregulates

431

immune-related genes in shrimp. Fish Shellfish Immunol. 32, 711–715. Bruno, G.G, Lucia, T.M., Ana, R., James, F.T., Valerie, I., Ana, L., Guerra, F., 1998. Species of

433

Vibrio isolated from hepatopancreas, haemolymph and digestive tract of a population of

434

healthy juvenile Penaeus vannamei. Aquaculture. 163,1–9.

437 438 439 440

Trop. Biomed. 2, 496–502.

Rosa, R.D., Barracco, M.A., 2010. Antimicrobial peptides in crustaceans. Invert Surviv J. 7(2),

SC

436

Fredrick, W.S., Ravichandran, S., 2012. Hemolymph proteins in marine crustaceans. Asian Pac. J.

262-284.

Wang, X.W., Wang, J.X., 2013. Diversity and multiple functions of lectins in shrimp immunity. Dev. Comp. Immunol. 39, 27–38.

M AN U

435

RI PT

432

441

Wang, X.W., Xu, J.D., Zhao, X.F., Vasta, G.R., Wang, J.X., 2014. A Shrimp C-type Lectin Inhibits

442

Proliferation of the Hemolymph Microbiota by Maintaining the Expression of Antimicrobial

443

Peptides. J. Biol. Chem. 289, 11779-11790.

445

Kaizu, A., Fagutao, F.F., Kondo, H., Aoki, T., Hirono, I., 2011. Functional Analysis of C-type

TE D

444

Lysozyme in Penaeid Shrimp. J. Biol. Chem. 286, 44344-44349. Fagutao, F.F., Koyama, T., Kaizu, A., Saito-Taki, T., Kondo, H., Aoki, T., Hirono, I., 2009.

447

Increased bacterial load in shrimp hemolymph in the absence of prophenoloxidase. FEBS. J.

448

276, 5298-5306.

450 451 452

Wang, X.W., Wang, J.X., 2015. Crustacean hemolymph microbiota: Endemic, tightly controlled,

AC C

449

EP

446

and utilization expectable. Mol. Immunol. 68, 404-411.

Ha, E.M., 2005. A Direct Role for Dual Oxidase in Drosophila Gut Immunity. Science. 310, 847-850.

453

Salathe, M., Holderby, M., Forteza, R., Abraham,W.M., Wanner, A., Conner, G.E., 1997. Isolation

454

and characterization of a peroxidase from the airway. Am. J. Respir. Cell Mol. Biol. 17,

455

97-105.

456

Kawahara, T., Quinn, M.T., Lambeth, J.D., 2007. Molecular evolution of the reactive

457

oxygen-generating NADPH oxidase (Nox/Duox) family of enzymes. BMC Evol. Biol. 7, 18

ACCEPTED MANUSCRIPT 458 459 460

109. Ryu, J.H., Ha, E.M., Lee, W.J., 2010. Innate immunity and gut-microbe mutualism in Drosophila. Dev. Comp. Immunol. 34, 369-376. De D.X., Corvilain, B., Dumont, J.E., Miot, F., 2014. Roles of DUOX-mediated hydrogen

462

peroxide in metabolism, host defense, and signaling. Antioxid. Redox Signal. 20, 2776-2793.

463

De, D.X., Wang, D., Many, M.C., Costagliola, S., Libert, F., Vassart, G., 2000. Cloning of two

464

human thyroid cDNAS encoding new members of the NADPH oxidase family. J. Biol. Chem.

465

275(30), 23227-23233.

RI PT

461

Xie, C.P., Chen, Y.P., Sun,W.W., Ding, J., Zhou, L.Z., Wang, S.S., Wang, S.Q., Zhang, Y.L., Zhu,

467

D.S., Wen, X.B., Hu, S.N., Li, S.K., 2014. Transcriptome and expression profiling analysis of

468

the hemocytes reveals a large number of immune-related genes in mud crab Scylla

469

paramamosain during Vibrio parahaemolyticus infection. PLoS One. 9 (12), e114500

471

M AN U

470

SC

466

Ohye, H., Sugawara, M., 2010. Dual oxidase, hydrogen peroxide and thyroid diseases. Exp. Biol. Med. (Maywood). 235, 424-433.

Corinne, D., Renée, O., Alexander,V., MarieSophie, N., Danielle. D., Alain. V., 1999. Purification

473

of a novel flavoprotein involved in the thyroid NADPH oxidase. cloning of the porcine and

474

human cDNAS. J. Biol. Chem. 274(52), 37265-37269.

TE D

472

Edens, W.A., Lisa, S., Cheng, G., Raymond, S., Kinkade, J.M., Taehoon, L., 2001. Tyrosine

476

cross-linking of extracellular matrix is catalyzed by duox, a multidomain oxidase/peroxidase

477

with homology to the phagocyte oxidase subunit gp91phox. J. Biol. Chem. 154(4), 879-892.

479 480 481 482 483 484 485 486 487

Furtmüller, P.G., Zederbauer, M., Jantschko, W., Helm, J., Bogner, M., Jakopitsch, C., 2006.

AC C

478

EP

475

Active site structure and catalytic mechanisms of human peroxidases. Arch. Biochem. Biophy. 445(2), 199-213.

Sumimoto, H., Miyano, K., Takeya, R., 2005. Molecular composition and regulation of the nox family NAD(P)H oxidases. Biochem Biophy Res Commun. 338(1), 677-686.

Guichard, C., Pedruzzi, E., Fay, M., Ben, M.S., Coant, N., Daniel, F., 2006. The nox/duox family of ROS-generating NADPH oxidases. Med Sci. 22(11), 953-959. Sommer, F., Backhed, F., 2015. The gut microbiota engages different signaling pathways to induce Duox2 expression in the ileum and colon epithelium. Mucosal Immunol. 8, 372-379. Sasakura, H., Moribe, H., Nakano, M., Ikemoto, K., Takeuchi, K., Mori, I., 2017. Lifespan 19

ACCEPTED MANUSCRIPT 488

extension by peroxidase and dual oxidase-mediated ROS signaling through pyrroloquinoline

489

quinone in C. elegans. J. Cell. Sci. 130, 2631-2643. Leto T.L., Morand, S., Hurt, D., Ueyama, T., Thomas, L., 2009. Targeting and regulation of

491

reactive oxygen species generation by nox family NADPH oxidases. Antioxid. Redox

492

Signal. 11(10), 2607-2619.

RI PT

490

493

Kumar, S., Molina-Cruz, A., Gupta, L., Rodrigues, J., Barillas-Mury, C., 2010. A peroxidase /dual

494

oxidase system modulates midgut epithelial immunity in Anopheles gambiae. Science. 327,

495

1644-1648.

Yang H.T., Yang M.C., Sun J.J., Shi X.Z., Zhao X.F., Wang J.X., 2016. Dual oxidases participate

497

in the regulation of intestinal microbiotic homeostasis in the kuruma shrimp Marsupenaeus

498

japonicus. Dev. Comp. Immunol. 59, 153-163.

M AN U

SC

496

499

Li, Y.Y., Xia, X.A.,Wu, Q.Y., Liu, W.H., Lin, Y.S., 2008. Infection with Hematodinium sp. in mud

500

crabs Scylla serrata cultured in low salinity water in southern China. Dis. Aquat. Org. 82,

501

145-150.

Juhasz, A., Ge, Y., Markel, S., Chiu, A., Matsumoto, L., Van, B.J., Roy, K., Doroshow, J.H., 2009.

503

Expression of NADPH oxidase homologues and accessory genes in human cancer cell lines,

504

tumours and adjacent normal tissues. Free Radic. Res. 43, 523-532.

TE D

502

Oliveira, P.L., Hu, X., Yang, R., Zhang, X., Chen, L., Xiang, X., Gong, C., Wu, X., 2013.

506

Molecular Cloning and Functional Characterization of the Dual Oxidase (BmDuox) Gene

507

from the Silkworm Bombyx mori. PLoS One. 8, e70118.

509 510 511

Inada, M., Kihara, K., Kono, T., Sudhakaran, R., Mekata, T., Sakai, M., Yoshida, T., Itami, T.,

AC C

508

EP

505

2013. Deciphering of the Dual oxidase (Nox family) gene from kuruma shrimp, Marsupenaeus japonicus: full-length cDNA cloning and characterization. Fish Shellfish Immunol. 34, 471-485.

512

Muñoz, M., Cedeño, R., Rodríguez, J., Knaap, W.P.W., Mialhe, E., Bachère, E., 2000.

513

Measurement of reactive oxygen intermediate production in haemocytes of the penaeid

514

shrimp, Penaeus Õannamei. Aquaculture. 191(1), 89-107.

515

Dunnill, C., Patton, T., Brennan, J., Barrett, J., Dryden, M., Cooke, J., 2015. Reactive oxygen

516

species (ROS) and wound healing: the functional role of ROS and emerging ROS-modulating

517

technologies for augmentation of the healing process. Int Wound. J, 14(1), 89. 20

ACCEPTED MANUSCRIPT 518

Grasberger, H., Gao, J., Nagao-Kitamoto, H., Kitamoto, S., Zhang, M., Kamada, N., Eaton, K.A.,

519

El-Zaatari, M., Shreiner, A.B., Merchant, J.L., Owyang, C., Kao, J.Y., 2015. Increased

520

expression of DUOX2 is an epithelial response to mucosal dysbiosis required for immune

521

homeostasis in mouse intestine. Gastroenterology. 149, 1849-1859. Koff, J.L., Shao, M.X., Ueki, I.F., Nadel, J.A., 2008. Multiple TLRs activate EGFR via a signaling

523

cascade to produce innate immune responses in airway epithelium. Am. J. Physiol. Lung Cell

524

Mol. Physiol. 294(6), L1068.

RI PT

522

Linderholm, A.L., Onitsuka, J., Xu, C., Chiu, M., Lee, W.M., Harper, R..W., 2010. All-trans

526

retinoic acid mediates duox2 expression and function in respiratory tract epithelium. Am. J.

527

Physiol. Lung Cell Mol. Physiol. 299(2), 215-21.

529

Cerenius, L., Söderhäll, K., The prophenoloxidase-activating system in invertebrates. Immunol Rev. 2004, 77(1):116-126.

M AN U

528

SC

525

530

Moskwa, P., Lorentzen, D., Excoffon, K.J., Zabner, J., McCray, P.B., Nauseef, W.M., 2007. A

531

novel host defense system of airways is defective in cystic fibrosis. Am. J. Resp. Crit

532

Care. 175(9), 174.

534

O'Neill, S., Brault, J., Stasia, M.J., Knaus, U.G., 2015. Genetic disorders coupled to ROS

TE D

533

deficiency. Redox Biol. 6, 135-156.

Zhang, Z., Li, S., Xie, C., Zhou, L., Li, C., Liu, W., Wen, X., 2015. Innate immune response and

536

gene expression of Scylla paramamosain under Vibrio parahaemolyticus infection. Aquac

537

Res. 46, 462-471.

539 540

Zhang, X.X., Sun, Z.Q., Zhang, X.X., Zhang, M., Li, S.K., 2018. Hemolymph Microbiomes of

AC C

538

EP

535

Three Aquatic Invertebrates as Revealed by a New Cell Extraction Method. Appl. Environ. Microb. 84, e02824-02817.

541

Givens, C.E., Burnett, K.G., Burnett, L.E., Hollibaugh, J.T., 2013. Microbial communities of the

542

carapace, gut, and hemolymph of the Atlantic blue crab, Callinectes sapidus. Marine Biology.

543

160, 2841-2851.

544

Longeon, A., Peduzzi, J., Barthelemy, M., Corre, S., Nicolas, J.L., Guyot, M., 2004. Purification

545

and partial identification of novel antimicrobial protein from marine bacterium

546

Pseudoalteromonas, species strain x153. Mar Biotechnol. 6(6), 633-641.

547

Williams, S.L., Jensen, R.V., Kuhn, D.D., Stevens, A.M., 2017. Analyzing the metabolic 21

ACCEPTED MANUSCRIPT 548

capabilities of a vibrio parahaemolyticus, strain that causes early mortality syndrome in

549

shrimp. Aquaculture. 476,44-48 Carolin, C., Wendling, F.M., Batista, K., Mathias. W., 2014. Persistence, seasonal dynamics and

551

pathogenic potential of Vibrio communities from Pacific oyster hemolymph. Plos One. 9(4),

552

e94256.

RI PT

550

553

Lafferty, K.D., Harvell, C.D., Conrad, J.M., Friedman, C.S., Kent, M.L., Kuris, A.M., 2015.

554

Infectious diseases affect marine fisheries and aquaculture economics. Annu Rev Mar Sci.

555

7(1), 471.

Xiong, W., Sun, Y., Zhang, T., Ding, X., Li, Y., Wang, M., 2015. Antibiotics, antibiotic resistance

557

genes, and bacterial community composition in fresh water aquaculture environment in

558

china. Microb Ecol. 70(2), 425-432.

M AN U

SC

556

559

Fečkaninová, A., Koščová, J., Mudroňová, D., Popelka, P., Toropilová, J., 2017. The use of

560

probiotic bacteria against Aeromonas infections in salmonid aquaculture. Aquaculture 469,

561

1-8.

Anson, K. K. Wu1., Andrew, M. K., John, S. Lumsden1., Brian, D., Janet, I. M., 2015. Complete

563

genome sequence of the fish pathogen Flavobacterium psychrophilum ATCC 49418. Stand

564

Genomic Sci. 10 (1) :3

TE D

562

Burioli, E.A.V., Varello, K., Trancart, S., Bozzetta, E., Gorla, A., Prearo, M., 2018. First

566

description of a mortality event in adult Pacific oysters in italy associated with infection by a

567

Tenacibaculum soleae strain. J. Fish. Dis. 41(2), 215-221.

569

Collado, L., Jara, R., Vásquez, N., Telsaint, C.,2014. Antimicrobial resistance and virulence genes

AC C

568

EP

565

of arcobacter, isolates recovered from edible bivalve molluscs. Food Control. 46, 508-512.

570

Desriac, F., Chevalier, P. L., Brillet, B., Leguerinel, I., Thuillier, B., Paillard, C., 2014. Exploring

571

the hologenome concept in marine bivalvia: haemolymph microbiota as a pertinent source of

572

probiotics for aquaculture. Fems. Microbiol. Lett. 350(1), 107-116.

573

22

ACCEPTED MANUSCRIPT

575

Table 1. Primers used in this study Sequence (5’-3’)

Objection

UPM (long)

CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT

RACE-PCR

UPM (short)

CTAATACGACTCACTATAGGGC

RACE-PCR

NUP

AAGCAGTGGTATCAACGCAGAGT

RACE-PCR

DUOX1-GSP3-1

CCTGGTGGGACGCCAACGACTTC

3’RACE

DUOX1-GSP3-2

GTATGCTGCACAGCCCGATCTGGTTA

DUOX1-GSP5-1

CCTGGGGCTGTTAGGGCTCTGGC

DUOX1-GSP5-2

GGGGTTCTGGTTGGTCCGCTGGT

DUOX2-GSP3-1

GAGTTTCACCGCTACATGGCGTCC

DUOX2-GSP3-2

CTGCCTCCGCCATGATGTTCACTTAC

M13F

CGCCAGGGTTTTCCCAGTCACGAC

M13R

AGCGGATAACAATTTCACACAGGA

Q_DUOX1F

GGATGTGATTCTCTGTAACGACG

qRT-PCR

Q_DUOX1R

TTCCTCACCGTGTTGTAGTCC

qRT-PCR

Q_DUOX2F

TGGAGACAAAATAGGCAACGAA

qRT-PCR

Q_DUOX2R

CCCGTGGTCCACAAAAACAGTA

qRT-PCR

siDUOX1F

GATCACTAATACGACTCACTATAGGGGCAAGACGTGTTCTTCCATTT

RNAi

siDUOX1R

AAGCAAGACGTGTTCTTCCATCCCTATAGTGAGTCGTATTAGTGATC

RNAi

siDUOX2F

GATCACTAATACGACTCACTATAGGGCCAAAGTACTTGCGCACAATT

RNAi

siDUOX2R

AACCAAAGTACTTGCGCACAACCCTATAGTGAGTCGTATTAGTGATC

RNAi

siGFP-F

GATCACTAATACGACTCACTATAGGGGGCTACGTCCAGGAGCGCACCTT

RNAi

siGFP-R

AAGGTGCGCTCCTGGACGTAGCCCCCTATAGTGAGTCGTATTAGTGATC

RNAi

β-actin-F

GCGGCAGTGGTCATCTCCT

qRT-PCR

β-actin-R

GCCCTTCCTCACGCTATCCT

qRT-PCR

27F

AGAGTTTGATCCTGGCTCAG

16S rRNA

1492R

TACGGTTACCTTGTTACGACTT

16S rRNA

RI PT

Primer

3’RACE 5’RACE 5’RACE

EP

TE D

M AN U

SC

3’RACE

AC C

574

23

3’RACE PCR screening PCR screening

ACCEPTED MANUSCRIPT Table 2. Basic information of mud crab hemolymph microbiota Miseq sequencing results Total_tag

Tax_tag

Uniq_tag

Average Length

OTUs number

DUOX2-1

79703

60481

19222

252

2156

DUOX2-2

75158

56036

19122

253

1090

DUOX2-3

80131

64484

15647

253

1848

DUOX2-4

80071

67460

12611

253

DUOX2-5

62116

53796

8320

252

GFP1

94740

80288

14452

253

GFP2

62876

57143

5733

252

GFP3

47004

34154

12850

GFP4

80208

65905

14303

GFP5

75730

66312

9418

AC C

EP

TE D

577

SC

RI PT

Sample

24

1068 779

1205 489

253

1347

253

1850

253

1568

M AN U

576

ACCEPTED MANUSCRIPT 578

Table 3. Matastat analysis of the relative abundances of hemolymph microbiota for the

579

siGFP and siDUOX2 groups at the genus level. Taxonomy

siGFP

P-value

Q-value

4.08%

0.07%

34.20%

0.41%

0.39%

75.80%

0.41%

4.15%

75.80%

0.24%

1.64%

75.80%

0.20%

3.43%

75.80%

Variance

S.D.

Mean

Variance

S.D.

Others

65.30%

0.06%

1.09%

48.68%

0.83%

Marinomonas

0.15%

0.00%

0.06%

1.37%

0.01%

Pseudoalteromonas

0.31%

0.00%

0.07%

1.08%

0.01%

Hydrogenophaga

0.13%

0.00%

0.04%

0.67%

0.00%

Shewanella

0.22%

0.00%

0.07%

0.62%

0.00%

Rubrobacter

0.15%

0.00%

0.04%

0.47%

Arenibacter

0.06%

0.00%

0.03%

0.37%

Altererythrobacter

0.05%

0.00%

Peredibacter

0.02%

0.00%

Aureispira

0.01%

0.00%

Bizionia

0.04%

0.00%

Haliangium

0.02%

0.00%

Thalassotalea

0.02%

0.00%

Megasphaera

0.01%

Phyllobacterium Burkholderiaceae

SC

RI PT

Mean (%)

0.00%

0.12%

0.84%

75.80%

0.00%

0.16%

3.77%

75.80%

M AN U

at genus level

siDUOX

0.24%

0.00%

0.09%

2.91%

75.80%

0.01%

0.21%

0.00%

0.09%

2.95%

75.80%

0.01%

0.14%

0.00%

0.07%

4.71%

75.80%

0.02%

0.13%

0.00%

0.03%

2.57%

75.80%

0.01%

0.07%

0.00%

0.03%

4.09%

75.80%

0.01%

0.07%

0.00%

0.02%

2.90%

75.80%

0.00%

0.01%

0.06%

0.00%

0.02%

2.87%

75.80%

0.01%

0.00%

0.01%

0.05%

0.00%

0.01%

0.10%

34.20%

0.09%

0.00%

0.02%

0.04%

0.00%

0.02%

2.13%

75.80%

0.07%

0.00%

0.02%

0.03%

0.00%

0.01%

2.37%

75.80%

0.00%

0.00%

0.00%

0.03%

0.00%

0.01%

3.65%

75.80%

Cellulophaga

0.00%

0.00%

0.00%

0.02%

0.00%

0.01%

2.44%

75.80%

Alkanindiges

0.09%

0.00%

0.03%

0.00%

0.00%

0.00%

0.72%

75.80%

Ferruginibacter

0.02%

0.00%

0.01%

0.00%

0.00%

0.00%

2.17%

75.80%

AC C

Aeromicrobium

EP

Allobaculum

TE D

0.02%

580 581

25

ACCEPTED MANUSCRIPT Figure legends

583

Fig. 1. Phylogenetic tree analysis of SpDUOXs relative to other known DUOXs proteins. 1000

584

bootstraps were performed on the Neighbor-Joining (NJ) tree to check repeatability of the results

585

using the MEGA 5.1 software, and the SpDUOXs is marked by black triangle ( ). Sp, Scylla

586

paramamosain; Mj, Marsupenaeus japonicus; Hs, Homo sapiens; Ce, Caenorhabditis elegans;

587

Cc ,Ceratitis capitata; Ld, Leptinotarsa decemlineata; Ot, Onthophagus taurus; Ac, Apis cerana;

588

Do, Drosophila obscura; As, Anopheles stephensi; Mi, Meloidogyne incognita; Nl,Nilaparvata

589

lugens; Pr,Pieris rapae; Zn, Zootermopsis nevadensis; Ar,Athalia rosae; Pg, Pseudomyrmex

590

gracilis; Cf, Camponotus floridanus; Dn, Dufourea novaeangliae; Mz, Maylandia zebra; Sd,

591

Seriola dumerili; Nm, Numida meleagris; Cl, Columba livia; Am, Alligator mississippiensis; Cm,

592

Chelonia mydas; Ol, Oryzias latipes; Cg, Crassostrea gigas; My, Mizuhopecten yessoensis.

M AN U

SC

RI PT

582

593

Fig. 2. Tissue distribution and expression patterns of SpDUOX1 and SpDUOX2. SpDUOX1

595

(A) and SpDUOX2 (B) transcript levels in various tissues were normalized to that in hemocytes,

596

and β-actin was used as the reference gene. HE: hemocytes; MI: mid-intestine; HP:

597

hepatopancreas; MU: muscle; ST: stomach; SE: subcuticular epidermis; GI: gill; HT: heart.

598

Expression profiles of SpDUOXs in hemocytes (C and D), and hepatopancreas (E and F) of mud

599

crabs after Vibro parahemolyticus and LPS challenges. (G) The levels of H2O2 during immune

600

challenges. Mud crabs injected with phosphate buffered saline (PBS) were used as control and

601

β-actin as the reference gene. Data is shown as mean ± S.D. Significance was compared between

602

the experimental and control groups at the same time point. Asterisks indicate the significant

603

differences (*P<0.05 and **P<0.01).

EP

AC C

604

TE D

594

605

Fig. 3. Bacterial count and ROS assay in hemolymph after SpDUOXs knockdown in mud

606

crab. The ROS levels and bacterial count in the hemolymph of mud crabs after RNA interference

607

(RNAi). RNAi experiments were conducted with the injection of specific DUOXs-siRNA or

608

negative GFP-siRNA. qRT-PCR was used to analyze RNA interference efficiency of (A)

609

SpDUOX1 and (B) SpDUOX2. The ROS in the mud crab hemolymph (C) and the hemolymph

610

bacterial count (D) was detected at 24, 36, 48 hours after gene silence of SpDUOX1, SpDUOX2,

611

and SpDUOX1+X2, respectively. β-actin was used as a reference gene for internal control. Data is 26

ACCEPTED MANUSCRIPT 612

shown as mean ± S.D. Significance was compared between the siRNA injection and the negative

613

groups at the same time point. Asterisks indicate the significant differences (*P<0.05 and

614

**P<0.01).

615 Fig. 4. In vivo bacterial clearance assay at 24 h post SpDUOXs RNA interference.

617

Hemolymph, withdrawn from different mud crabs treated with DEPC, siGFP, siSpDUOX1 and

618

siSpDUOX2 followed by injection with V. parahaemolyticus, was diluted with 0.8% NaCl (1/20)

619

and cultured on a 2216E solid plate at 37 °C for 18 h. The number of colonies was counted and

620

recorded. GFP-siRNA was used as negative group and DEPC as a blank group. Asterisks indicated

621

significant differences (*P<0.05 and **P<0.01).

M AN U

622

SC

RI PT

616

Fig. 5. Bacterial community composition in the hemolymph of mud crab after SpDUOX2

624

RNA interference. Non-Metric Multi-Dimensional Scaling (NMDS) analysis. (A), Principal

625

Component analysis (PCoA) (B) and Shannon index analysis (C). Relative abundance of the top

626

10 phylum (D) and the top 20 genus (E). Venn diagram of the relative abundances of hemolymph

627

microbiota between the siGFP and siDUOX2 groups at the genus level (F).

AC C

EP

TE D

623

27

628 Fig. 1

AC C

629

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

28

ACCEPTED MANUSCRIPT B

Relative expression of SpDUOX2

Relative expression of SpDUOX1

400

** ** 200

**

**

8

** 4

*

0

HE

MU

SK

GI

MI

HT

6

** 4

2

RI PT

A

0

HP

MU

GI

SK

630 D * 8

LPS V. parahemolyticus PBS

4

*

2

0 0h

6h

12h

24h

48h

Time post injection

Hepatopancreas

8

6

LPS V.parahemolyticus PBS

EP

*

2

*

**

*

0

0h

6h

12h

24h

48h

96h

HP

LPS V. parahemolyticus PBS

1.0

* *

0.5

0.0

F

6h

5

12h

24h

Hepatopancreas

4

48h

96h

*

3

*

2

1

*

0 6h

12h

24h

Time post injection

29

LPS V.parahemolyticus PBS

*

0h

Time post injection

632

1.5

0h

**

4

Hemocytes

* **

2.0

96h

TE D

**

AC C

Ralative expression of SpDUOX1

10

MI

Time post injection

631 E

2.5

M AN U

6

*

HT

SC

Hemocytes

Relative expression of SpDUOX2

10

Relative expression of SpDUOX2

Relative expression of SpDUOX1

C

HE

Tissue type

Tissue type

48h

96h

ACCEPTED MANUSCRIPT *

PBS V.parahemolyticus

80

60

40

RI PT

The content of H2O2(mmol/L)

G 100

20

0 0h

6h

12h

24h

48h

96h

Time post injection

633

EP

TE D

M AN U

SC

Fig. 2

AC C

634 635

30

ACCEPTED MANUSCRIPT 636 B

1.0

*

0.5

**

0.0 24h

36h

2.0

siGFP siDUOX 1.5

*

1.0

RI PT

expression of SpDUOX2

siGFP siDUOX

Relative

Relative expression of SpDUOX1

A 1.5

** 0.5

0.0

48h

24h

Time post siRNA treatment

36h

*

**

**

*

*

*

30

24h

36h

48h

15

siGFP siDUOX1 siDUOX2 siDUOX1+2

** **

10

**

*

5

24h

36h

Time post siRNA treatment

AC C

EP

TE D

Time post siRNA treatment

Fig. 3.

**

0

0

638 639

**

M AN U

The content of H2O2(mmol/L)

90

60

The number of microbiata in the hemolymph(104)

D 20 siGFP siDUOX1 siDUOX2 siDUOX1+2

SC

637 C120

48h

Time post siRNA treatment

31

48h

ACCEPTED MANUSCRIPT

640

641

642

643

SC M AN U

** **

1 X O U siD

TE D

NS

FP siG 2 X O U siD

EP

C EP D

32

AC C

10

5

0

15

RI PT

Fig. 4.

The number of bacteria in 3 the hemolymph(10 CFU/ml)

ACCEPTED MANUSCRIPT 644 A 0.30

B siGFP siDUOX

siGFP4

siGFP1

0.15

siGFP siDUOX

siDUOX4

0.2 siDUOX3 siDUOX5

siGFP4

siDUOX5

MDS2

siDUOX2

siGFP2 siGFP3

0.0

siGFP5

-0.2

-0.15

siDUOX2

siGFP2siGFP3

RI PT

siDUOX3

0.00

PCoA2(19.29%)

siDUOX4 siGFP1 siGFP5

siDUOX1

siDUOX1

-0.30

-0.4

-0.4

-0.2

0.0

0.2

-0.4

0.4

-0.2

0.0

0.2

PCoA1(49.4%)

MDS1(stress: 0.063)

M AN U

SC

645

647

AC C

EP

TE D

646

33

0.4

RI PT

ACCEPTED MANUSCRIPT

M AN U

SC

648

649

EP

TE D

Fig.5

AC C

650

34

ACCEPTED MANUSCRIPT Dear editor, The highlights are listed as following: ① full-length of SpDUOX1 with 4786 bp and SpDUOX2 with 5093 bp was isolated

RI PT

from mud crab. ② SpDUOX1 and SpDUOX2 might participate in maintaining bacterial homeostasis in the hemolymph of mud crabs.

M AN U

bacterial community composition.

SC

③ The crabs with reduced SpDUOX2 gene expression showed a profound difference in

With my best wishes!

AC C

EP

TE D

Shengkang LI