Evolutionary and functional analysis of MyD88 genes in pearl oyster Pinctada fucata martensii

Journal Pre-proof Evolutionary and functional analysis of MyD88 genes in pearl oyster Pinctada fucata martensii Yu Jiao, Zefeng Gu, Shaojie Luo, Yuewen Deng PII:

S1050-4648(20)30096-6

DOI:

https://doi.org/10.1016/j.fsi.2020.02.018

Reference:

YFSIM 6817

To appear in:

Fish and Shellfish Immunology

Received Date: 4 November 2019 Revised Date:

5 February 2020

Accepted Date: 10 February 2020

Please cite this article as: Jiao Y, Gu Z, Luo S, Deng Y, Evolutionary and functional analysis of MyD88 genes in pearl oyster Pinctada fucata martensii, Fish and Shellfish Immunology (2020), doi: https:// doi.org/10.1016/j.fsi.2020.02.018. 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. © 2020 Published by Elsevier Ltd.

1

Evolutionary and functional analysis of MyD88 genes in

2

pearl oyster Pinctada fucata martensii

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Yu Jiao1, 2, Zefeng Gu1, Shaojie Luo1, Yuewen Deng, 1, 2

5

1. Fishery College, Guangdong Ocean University, Zhanjiang 524025, China;

6

2. Guangdong Technology Research Center for pearl aquaculture and process, Zhanjiang

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524025, China;

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 Address correspondence to: [email protected]

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Abstract:

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Myeloid differentiation factor 88 (MyD88) is an adapter protein that links toll-like

12

receptors and interleukin 1 receptor-mediated signal transduction. In this study, we

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identified 20 MyD88 genes from eight mollusk genomes and found that MyD88 was

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expanded in bivalves. This expansion tends to be tandem duplication. Phylogenetic

15

analysis suggested that the tandem duplication of MyD88 was formed before bivalve

16

differentiation. All of the identified MyD88 contained both of death domain (DD) and

17

toll/interleukin-1 receptor (TIR) domain, and 13 mollusks MyD88 have low

18

complexity regions (LCRs), which was not found in the MyD88 gene from humans

19

and zebrafish. The genomic structure showed that most of the mollusk MyD88 (14 of

20

19) contained five conserved introns, four of which were found in humans and

21

zebrafish. Furthermore, the cDNA full length of PfmMyD88-2 (one of the two

22

identified MyD88 in Pincatada fucata martensii) was obtained with 1591 bp,

23

including 260 bp of 5ʹUTR, 257 bp of 3ʹUTR, and 1077 bp of open reading frame

24

encoding 358 amino acids. Quantitative real-time PCR analysis demonstrated that

25

PfmMyD88-2 mRNA was widely expressed in all detected tissues. The highest

26

expression level was in the gills and followed by hepatopancreas and feet. After 1

27

lipopolysaccharide stimulation, PfmMyD88-2 expression level increased and reached

28

the highest level at 12 h and then gradually declined to the normal level.

29

Over-expression of PfmMyD88-2 in HEK293T increased the luciferase activity of the

30

pNF-κB-Luc reporter. We also identified that PfmmiR-4047 could regulate the

31

expression of PfmMyD88-2. These results help us elucidate the mechanism

32

underlying mollusk immune response.

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Keywords: Pinctada fucata martensii; MyD88; mollusk; immune Response;

34 35

1. Introduction

36

The innate immune system is the first line of defense of an organism against

37

invading pathogens. To cope with pathogenic microorganisms, invertebrates rely

38

solely on their innate immune system because they do not have adaptive immunity [1].

39

The innate immune system is triggered by various pattern recognition receptors

40

(PRRs) that can recognize pathogen-associated molecular patterns (PAMPs) [2].

41

Toll-like receptors (TLRs), as one of the major types of PRRs, play a key role in

42

innate immunity by recognizing PAMPs, such as lipopolysaccharides (LPS),

43

peptidoglycans, polyinosinic–cytidylic acid, β-glycan of fungi, and lipoproteins of

44

various pathogens [3]. Myeloid differentiation factor 88 (MyD88) is a key adapter

45

protein of the TLR signal pathway, and it mediates all the signal pathways except

46

TLR3. After the recognition of PAMPs by TLRs, MyD88 could recruit TLR through

47

one of its conserved domains, toll/interleukin-1 receptor (TIR). And then, MyD88

48

uses its other conserved death domain (DD) to associate with the DD of interleukin-1

49

receptor-associated kinase, and triggers the activation of nuclear factor-kappa B

50

(NF-κB) and mitogen-activated protein kinase pathways [4].

2

51

MyD88 genes are ubiquitous and conserved in the animal kingdom.

52

MyD88-mediated signaling pathways play crucial roles in the immune response of

53

invertebrates. Their connections with pathogen challenge (LPS or virus) were also

54

investigated [5].

55

been identified in many species, such as Danio rerio [6], Crassostrea gigas [7],

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Chlamys farreri [8], Ruditapes philippinarum [9], Scylla paramamosain [10], and

57

Rana dybowskii [11]. In contrast with insects and vertebrates that only have one copy

58

of MyD88 [12-14], many MyD88 genes have been identified in mollusks. A total of

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five MyD88 genes were found in the genome of Yesso scallop (Patinopecten

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yessoensis) [15], 10 were found in C. gigas [16], and three were identified in

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Mediterranean mussel (Mytilus galloprovincialis) [17]. Two MyD88 duplications

62

(HcMyD88-1 and HcMyD88-2) were found in the transcriptome of triangle-shell

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pearl mussel (Hyriopsis cumingii) [18]. These reports suggest that many copies of

64

MyD88 genes exist in the genomes of mollusks, but their evolution is still unclear. An

65

increasing number of genomes of mollusks have been sequenced and released,

66

thereby providing the basis for detailed analysis of MyD88 genes at the genomic

67

level.

Since the first discovery of MyD88 in 1990, MyD88 genes have

68

Pinctada fucata martensii is one of the main species cultured for marine pearl

69

production. This species is widely distributed in Guangdong, Guangxi, and Hainan in

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China. In recent years, with the continuous expansion of mariculture, coastal water

71

quality deteriorated, thereby increasing the number of various mollusk diseases and

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dramatically decreasing pearl production. Therefore, the mechanism underlying the

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immune response in pearl oysters must be determined [13]. One of the MyD88 genes

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in P. f. martensii has been identified and functionally characterized [19]. While, the

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regulatory mechanism of MyD88 gene expression in mollusk was not clear. MiRNAs 3

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are short non-coding RNAs that are direct negative regulators of gene expression by

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binding to specific sequences within a target mRNA[20]. MiRNAs are changing the

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way we think about the development of the immune system and regulation of immune

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functions. MiRNAs are implicated in establishing and maintaining the cell fate of

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immune cells [21], and they are involved in innate immunity by regulating immune

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signaling and ensuing cytokine response [22]. Previously, we have verified that

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miR-146 and miR-29a participated in the immune response of pearl oyster P. f.

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martensii [23, 24].

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In this paper, the genomic identification and characterization of MyD88 genes

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were performed on the published genome of various mollusk species, namely, P. f.

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martensii, C. gigas, Patinopecten yessoensis, Modiolus philippinarum, Bathymodiolus

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platifrons, Aplysia californica, Octopus bimaculoides, and Lottia gigantean, and two

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vertebrate genomes, namely, D. rerio and Homo sapiens. Furthermore, the unreported

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MyD88 gene from P. f. martensii (Pm-MyD88-2) was cloned and functionally

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identified, and one regulatory miRNA of Pm-MyD88-2 was obtained by target

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analysis and verification. These results could help to enhance our understanding of

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mollusk immune response.

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2. Materials and Methods

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2.1 Identification of MyD88

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The gene annotation information of P. f. martensii and C. gigas was obtained

96

from previous studies [25, 26]. The gene annotation information of M. yessoensis, M.

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philippinarum, B. platifrons, A. californica, O. bimaculoides, L. gigantean, D. rerio,

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and H. sapiens were downloaded from the National Center for Biotechnology

99

Information database. The genes annotated as MyD88 were screened, and the protein

100

domain of all the screened MyD88 genes was re-analyzed and confirmed by Simple 4

101

Modular

Architecture

Research

Tool

(SMART)

version

5.1

102

(http://smart.Embl-heidelberg.de/). Only the genes that were homologous with

103

MyD88 (E-value=1-e5) and contained both DD and TIR domains were used in the

104

analyses of this study.

105

2.2 Sequence analysis Based on amino acid sequences, comparison and phylogenetic analysis were

106 107

performed

108

(http://www.genome.jp/tools-bin/clustalw). Phylogenetic trees were constructed by

109

using MEGA7.0 with the neighbor-joining (NJ) algorithm. Confidence values were

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obtained with bootstrapping with 1000 replications [27]. Gene structure information

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for MyD88 was obtained from published genome data. The exon and intron structures

112

were

113

http://gsds.cbi.pku.edu.cn/). The target prediction between miRNAs and the 3’UTR of

114

MyD88 was performed using RNAhybrid. The miRNAs used in the target analysis

115

were from our previous study [28].

116

2.3 Experimental animals, RNA extraction, and cDNA synthesis

drew

with

using

ClustalW

the

Gene

multiple

Structure

sequence

Display

Server

alignment

(GSDS,

117

Experimental individuals were produced by the fifth generations of a

118

fast-growing group. Pearl oysters were cultured in the sea area of Xuwen County,

119

Zhanjiang City, Guangdong, China. Before the experiment, the samples were fed at

120

25 °C to 27 °C in the tank with recirculating filtered seawater for 3–5 days. The

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tissues, including adductor muscle, foot, gonad, mantle, hemocytes,

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hepatopancreas and gills, were removed from P. f. martensii and quickly stored in

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liquid nitrogen until use. Hemocytes were stored in a centrifuge tube containing trizol

124

after centrifugation. RNA (1 μg) was obtained according to the operating instructions.

125

RNA quality was measured by using a NanoDrop 2000 spectrophotometer. The 5

and

126

integrity of the RNA was determined by 1.0% agarose gel. The total RNA was used

127

as the template, and random primer and M-MLV reverse transcriptase were added to

128

obtain cDNA.

129

2.4 Rapid amplification of cDNA ends (RACE)

130

A total of two MyD88 genes (AMQ81593.1 and Pfm-10008089) were identified

131

from P. f. martensii. AMQ81593.1 has been cloned and functionally characterized

132

previously [19]. We cloned the full cDNA sequence of MyD88 (Pfm-1008089),

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named as PfmMyD88-2, and verified its function. The cDNA sequence of

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PfmMyD88-2 were obtained from the published genome [26]. The sequence was used

135

as a template, and Primer Premier 5.0 was used to design the full-length and real-time

136

PCR primers (Table 1). The templates were prepared according to the operating

137

instructions for the SMARTTM rapid amplification of cDNA ends (RACE).

138

Amplification system was incubated with 2 μL of cDNA, 12.5 μL of Premix LA Taq

139

Hot start, 1 μL of PfmMyD88-2-5ʹRACE-outer (10 μm) or 1 μL of

140

PfmMyD88-2-3ʹRACE-outer (10 μm), 1 μL of UPM (10 μm), and 8.5 μL of ddH2O.

141

In the secondary nested PCR, the first amplification product was used as a template

142

for

143

PfmMyD88-2-5ʹRACE-inner (10 μm) or PfmMyD88-2-3ʹRACE-inner (10 μm) and

144

NUP (10 μm). The purified PCR amplification product was subcloned into pMD-19T

145

vector and transformed into DH-5α.

146

2.5 Quantitative real-time PCR

the

second

time.

In

addition,

the

primer

was

replaced

by

147

Quantitative real-time PCR (qRT-PCR) was performed to detect the

148

PfmMyD88-2 expression in different tissues. The amplification system included 5 μL

149

of SYBR Premix ExTaqTM, 1 μL of primer, 0.5 μL of cDNA, and 3.5 μL of ddH2O.

150

PCR program included one cycle at 94 °C for 3 min, followed by 40 cycles at 94 °C 6

151

for 15 s, 58 °C for 15 s, 72 °C for 1 min, and a final single cycle at 72 °C for 10 min.

152

GAPDH was used as the internal reference gene [29].

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2.6 Lipopolysaccharide (LPS) stimulation

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A total of 120 healthy pearl oysters P. f. martensii were randomly divided into

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two groups: the experimental and control groups. Sixty individuals were included in

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each group. The experimental group was injected with 100 μL 10 μg/mL of LPS

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solution. The control group was injected with 100 μL phosphate buffer saline (PBS).

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Then, at diff erent times (0, 2, 4, 8, 12, 24, and 36 h) after the surgical implication,

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hemolymph from at least 8 host pearl oysters of each group were collected using 1mL

160

syringes from adductor muscles. The hemolymph was subjected to centrifugation at

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3500 r/min for 5 min and the precipitant at the bottom was separated to collect

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hemocytes. Then, the hemocytes immersed in TRIzol reagent (Invitrogen, USA), and

163

stored at −80°C. All samples were stored in trizol until use.

164

2.7 Vector construction, cell culture and transfection

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For expression plasmids, the open reading frame (ORF) fragment of

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PfmMyD88-2 was amplified with the primers listed in Table 1 and subcloned into

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pcDNA3.1 vector (Invitrogen, USA). The reporter plasmid pNF-κB-Luc was obtained

168

from Clontech (USA), and pRL-TK Renilla luciferase plasmid (Progema, USA) was

169

used as an internal control. The 3ʹUTR of MyD88 gene was amplified using PCR and

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cloned into the pMIR-REPORT vector between the Spe I and Hind III sites,

171

immediately downstream from the Renilla luciferase gene. The primers used for in

172

this study are listed in Table 1.

173

The procedure for target verification were conducted according to the method

174

described by Tian et al [23]. Briefly, HEK293T cells were cultured at 37 °C with

175

dulbecco modified eagle medium (DMEM) containing 10% fetal bovine serum in a 7

176

humidified incubator under 5% CO2. Before the plasmid transfection, cells were

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seeded into a 48-well culture plate, and the number of inoculated cells was 105 per

178

hole with 500 μL medium. To identify the function of PfmMyD88-2 in NF-κB

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pathway, pNF-κB-Luc and pcDNA3.1-PfmMyD88-2 or pcDNA3.1 vector was

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co-transfected into HEK293T cells using Lipofectamine™ 2000 (Invitrogen). For the

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miRNA

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(pMIR-REPORT-3ʹUTR/PfmMyD88-2)

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(CCAGACACUCAGAAACACGAUU, GenePharma, Shanghai, China) or negative

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control (NC, UCACAACCUCCUAGAAAGAGUAGA, GenePharma, Shanghai,

185

China) mimics were co-transfected into HEK293T cells using Lipofectamine™ 2000

186

(Invitrogen). At 24 h after the transfection, luciferase activity was measured using a

187

dual luciferase assay kit (Promega, Madison, WI, USA) according to the

188

manufacturer’s protocol. All assays were performed with three independent

189

transfections. MicroRNA and NC mimics were synthesized in Genepharma, Shanghai,

190

China, and diluted in 0.1 μg/μL with DEPC water.

191

2.8 Overexpression of PfmmiR-4047

target

verification

experiment, and

luciferase

reporters PfmmiR-4047

192

A total of 100 μL PfmmiR-4047 and NC mimic solutions were separately

193

injected into the muscle of P. f. martensii. After 24 h, hemocytes were collected from

194

different individuals and stored in trizol before use. Stem loop qRT-PCR was

195

performed to measure the expression levels of the putative target genes and miRNA.

196

2.9 Statistical Analysis

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The data were analyzed using one-way ANOVA in SPSS 22.0. A p-value less

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than 0.05 (p < 0.05) was considered as statistically significant

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3. Results

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3.1 Expansion of MyD88 in bivalves 8

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On the basis of the gene annotations combined with SMART analysis, we

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identified 17 MyD88 genes from five bivalve genomes, including 2 from P. f.

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martensii, 4 from C. gigas, 5 from M. yessoensis, 4 from M. philippinarum, and 2

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from B. platifrons (Table 2). Only one MyD88 gene was identified from A. californica,

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O. bimaculoides, L. gigantean, D. rerio, and H. sapiens. The gene number of MyD88

206

in bivalves was expanded, compared with that of other mollusks and vertebrates.

207

Among the identified MyD88 genes, we identified one tandem array of two and three

208

MyD88 genes from C. gigas and M. philippinarum, respectively. Two tandem arrays

209

of MyD88 were found in M. yessoensis. No tandem arrays of MyD88 were found in P.

210

f. martensii and B. platifrons.

211

3.2 Sequence analysis of the MyD88 family

212

SMART analysis showed that all of the MyD88 genes contained the conserved

213

DD and TIR domain. Apart from the conserved DD and TIR domains, 13 of the

214

identified 20 mollusk MyD88 genes have 1-4 low complexity regions (LCRs), but this

215

region was not found in humans and zebrafish (Table 2).

216

Phylogenetic tree analysis indicated that all of the MyD88 genes from the

217

mollusk were classified together and divided into two branches (Fig. 1), indicating the

218

expansion of MyD88 in bivalve was derived from the ancestral gene shared by

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mollusks. The tandem array of two MyD88 genes (Cgi-10026092 and 10026099)

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from C. gigas were separated into two branches. And a similar phenomenon was

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observed in M. philippinarum, Mph-11876-0.6 was separated from its tandem-linked

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Mph-11876-0.10 and Mph-11876-0.8. Two MyD88 genes from P. f. martensii

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(Pfm-10008089 and Pfm-AMQ81593.1) were located in different branches and

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clustered together with the separated two tandem-linked MyD88 (Cgi-10026092 and

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10026099) in C. gigas, respectively. These results indicated that the tandem 9

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duplication of MyD88 was formed before bivalve differentiation. During evolution,

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the sequence of MyD88 changed and separated gradually, and its function also

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diversified. In C. gigas, 10026092 and 10026099 have completely different

229

expression patterns during development; 10026092 has a relatively stable expression

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level after blastula stage, whereas 10026099 has higher expression level between

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Early umbo larva and umbo larva (Supplemental Fig. 1).

232

Then, we analyzed the genomic structure of 19 MyD88 genes except

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PmMyD88-1 as it has no genomic information. Results showed that most of the

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MyD88 genes (14 out of 19) from the mollusks contained five introns, whereas

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human and zebrafish contained four introns (Fig. 2). Detailed analysis revealed that

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the five intron positions (marked in purple in Fig. 3) were conserved among mollusks,

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four of which were also found and conserved in human and zebrafish. During

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evolution, some species obtained some species-specific intron (marked in yellow in

239

Fig. 3). For example, the MyD88 genes from P. f. martensii (Pfm-10008089) and C.

240

gigas (Cgi-10026092) have the same introns at similar positions, indicating that this

241

intron occurred before the separation of P. f. martensii and C. gigas.

242

3.3 Gene cloning of MyD88 (Pfm-10008089) from P. f. martensii

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A total of two MyD88 (Pfm-AMQ81593.1 and PfmMyD88-2) genes were

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identified from P. f. martensii. We cloned the full cDNA sequence of PfmMyD88-2

245

and verified its function. The full-length cDNA of PfmMyD88-2 was 1591 bp,

246

including 260 bp of 5ʹUTR, 257 bp of 3ʹUTR with 28 bp poly(A) tail, and a typical

247

polyadenylation signal (AATAAA), and 1077 bp of open reading frame encoding 358

248

amino acid (Fig. 4).

249

3.4 Expression of PfmMyD88-2 in tissues and after lipopolysaccharide

250

stimulation 10

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To understand the function of PfmMyD88-2, we analyzed its expression pattern

252

in different tissues, including adductor muscle, foot, gonad, hepatopancreas, mantle,

253

hemocytes, and gills from P. f. martensii. PfmMyD88-2 can be easily detected in all

254

tissues. It was highly expressed in the gills and hepatopancreas but lowly expressed in

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adductor muscle and hemocytes (Fig. 5a). After LPS stimulation, the expression of

256

PfmMyD88-2 in hemocytes increased and reached the maximum level at 12 h, after

257

which it was downregulated and returned to the normal level at 24 h (Fig. 5b).

258

3.5 PfmMyD88-2 was involved in nuclear factor-kappa B signal pathway

259

To determine the signal pathway after PfmMyD88-2 over-expression, we

260

recombined the ORF of PfmMyD88-2 into the pcDNA3.1 plasmid. The constructed

261

pcDNA3.1-PfmMyD88-2 and the empty vector pcDNA3.1 were co-transfected into

262

HEK293T cells with pNF-κB-Luc reporter, respectively. Dual-luciferase reporter

263

assays showed that the luciferase activity of the pNF-κB-Luc reporter co-transfected

264

with pcDNA3.1-PfmMyD88-2 was substantially increased by 1.94-fold compared

265

with the empty vector pcDNA3.1 (Fig. 6).

266

3.6 PfmMyD88-2 was regulated by PfmmiR-4047

267

Based on target prediction by RNAhybrid, PfmmiR-4047, one identified

268

conserved miRNA in our previous research [28], was predicted as the regulatory

269

miRNA of PfmMyD88-2. The target interaction between PfmmiR-4047 and

270

PfmMyD88-2 is shown in Fig. 7a. To verify that PfmMyD88-2 could be negatively

271

regulated by PfmmiR-4047, the luciferase reporter plasmid containing 3ʹUTR of the

272

PfmMyD88-2 gene was generated. The constructed reporter plasmid was transfected

273

into HEK293T cells with PfmmiR-4047 mimics or the control NC mimics. Following

274

24 h of incubation, the cells were subjected to luciferase assays. As shown in Fig. 7b,

275

PfmmiR-4047 mimics significantly reduced the luciferase activity of the reporter 11

276

containing 3ʹUTR of PfmMyD88-2 gene. After injection of PfmmiR-4047 mimics

277

into the pearl oysters, the expression of PfmmiR-4047 increased by 3.20 times and

278

5.32 times in the hemocytes compared with the NC and DEPC groups, respectively

279

(p<0.05) (Fig.7c). PfmMyD88-2 expression was significantly decreased to 90.16%

280

and 90.03% compared with the NC and DEPC groups, respectively (p<0.05) (Fig.

281

7d).

282

4. Discussion

283

Our analysis of the genomic data revealed an expansion of MyD88 in bivalve

284

mollusks compared with that in Gastropoda and vertebrates. Bivalve mollusks have

285

no adaptive immunity while they thrive in microbe-rich environments as filter-feeders.

286

Thus, bivalve mollusks, such as oysters, have developed remarkable tolerance to

287

biotic and abiotic stresses [25, 30]. The adaptation and resilience of bivalve mollusks

288

are supported by the expansion and diversity of many stress- and immune-related

289

genes [25, 30-34]. Expansion of TLR pathway has been described in oysters, thereby

290

providing preliminary evidence for highly specific functional responses to biotic

291

challenge by specific members of large multigene families encoding innate

292

immune-type molecules [16]. The expansion of MyD88 genes may enhance the

293

capacity of bivalve mollusks to handle and respond to invading pathogens.

294

Gene location and structure analysis indicated that the expansion of MyD88 in

295

bivalve mollusks occurred by tandem duplication. Local tandem duplication can be a

296

major mechanism of immune gene expansion in oyster; a large proportion of the

297

expanded innate repertoire exists as tandem gene clusters, which may be driven by

298

both biotic and abiotic stresses [16]. Gene duplication and expansion are important

299

sources of evolutionary novelty as selection maintains duplicated genes only through

300

functional divergence [35, 36]. After duplication, the function of MyD88 was 12

301

diversified and the two tandem-linked MyD88 from C. gigas may be endowed with

302

different functions during development as their different expression patterns, thereby

303

enhancing their capacity to cope with different invading pathogens. This expansion

304

was derived from the ancestral gene shared by mollusks, and then, it expanded in the

305

bivalve lineage.

306

Protein domain analysis showed all of the identified MyD88 genes in this study

307

contained the conserved DD and TIR domain. In this study, only 4 of the 10 reported

308

MyD88 from C. gigas [16] have both of DD and TIR domains and considered as

309

MyD88 genes. Interestingly, 13 of the identified 20 MyD88 genes from mollusks had

310

LCR, but not found in vertebrates. LCRs are regions of biased composition normally

311

consisting of a regular repeat, thereby providing abundant material for new functions

312

[37]. Additionally, these regions are not strongly conserved in length and evolve

313

rapidly, although many participate in crucial molecular functions [38]. Some types of

314

LCRs are usually found in proteins with particular functional classes, especially

315

transcription factors and protein kinases [39]. Compared with proteins without LCRs,

316

proteins containing LCRs tend to have more interactions with other proteins [39]. We

317

proposed that MyD88 in mollusks may combine with more receptors and mediate

318

other signal transductions, but this assumption requires further experimental

319

validation.

320

To analyze the role of MyD88 in the innate immune response of pearl oysters,

321

we cloned the full-length sequence of PfmMyD88-2 and verified its function. The

322

hemocytes of mollusks play crucial roles in the innate immune response. However, a

323

relatively low expression level was observed in the hemocytes, similar with that in

324

Hyriopsis cumingii [18]. We speculated that PfmMyD88-2 expression in hemocytes

325

may be induced in a stressed state. The temporal expression of PfmMyD88-2 after 13

326

LPS challenge in hemocytes was examined. LPS molecules are found in the outer

327

membrane of Gram-negative bacteria and functions as an endotoxin that elicits a

328

strong immune response in animals [40]. As the key signaling adaptor in the TLR

329

signal pathway, MyD88 can transmit the LPS signal to activate a transcription factor,

330

NF-κB, and other downstream kinases [41]. In P. f. martensii, PfmMyD88-2

331

increased significantly at 8–12 h after LPS treatment, thereby suggesting that

332

PfmMyD88-2 is involved in LPS signaling and thus plays a role in the innate immune

333

response of P. f. martensii. A previous study reported that the PfmMyD88

334

(Pfm-AMQ81593.1) was highly induced at 4 h after LPS stimulation [19]. The

335

different response times of PfmMyD88 suggest their different functions, indicating

336

that the function of the two PfmMyD88s has diversified.

337

The result of dual luciferase reporter assays indicated that PfmMyD88-2 could

338

activate the luciferase activity of the reporter gene pNF-κB-Luc in HEK293T cell,

339

thereby suggesting that PfmMyD88-2 is involved in NF-κB pathway, similar to

340

mammalian cells [41, 42]. In invertebrates, as the lack of adaptive immune system,

341

TLR pathway via NF-κB have been confirmed as the most important signal pathway

342

in the host defense [43]. Combined with the expression pattern of PfmMyD88-2 in

343

tissues and after LPS stimulation, we concluded that MyD88-dependent signaling

344

pathway via NF-κB plays an important role in innate immune response.

345

Furthermore, we identified one regulatory miRNA of PfmMyD88-2. An

346

increasing number of studies have provided evidence that miRNAs may be transferred

347

from one species to another and regulate gene expression in the recipients’ cells. The

348

most intriguing results revealed that stable miRNAs derived from food plants may

349

enter the mammals’ circulatory system and inhibited the production of specific

350

mammalian proteins after reaching the target [44]. Feeding with miRNA mimics or 14

351

inhibitors added to an artificial diet makes Myzus persicae nicotianae more or less

352

sensitive to the toxic effects of nicotine [45]. MiR-4047 was found in

353

Ciona intestinalis firstly, but its function was not clear. In this paper, we found that

354

PfmmiR-4047 could regulate the expression of PfmMyD88-2 and involved in the

355

immune response in pear oyster P. f. martensii. We proposed that oral feeding of

356

synthetic PfmmiR-4047 mimics may help regulate the immune response in pearl

357

oysters, but this proposal needs further research.

358

In conclusion, we present a compressive genome-wide identification of MyD88

359

genes in eight mollusk genomes, namely, P. f. martensii, C. gigas, M. yessoensis, M.

360

philippinarum, B. platifrons, A. californica, O. bimaculoides, L. gigantean, and two

361

vertebrate genomes, namely, D. rerio, and H. sapiens. MyD88 genes were expanded

362

in bivalves. Phylogenetic tree analysis showed that this expansion of MyD88 was

363

derived from the ancestral gene shared by mollusks, and then, it expanded in the

364

bivalve lineage. The genomic structure and domain combination indicated that

365

MyD88 genes are very conservative in invertebrates and vertebrates. We obtained the

366

cDNA sequence of PfmMyD88-2 from P. f. martensii and determined its function in

367

the immune response. It is involved in the NF-κb pathway and is regulated by

368

PfmmiR-4047.

369

Acknowledgments

370

The studies were financially supported by grants of the National Natural Science

371

Foundation of China (31672626), Innovation Team Project from the Department of

372

Education of Guangdong Province (Grant no. 2017KCXTD016), Modern Agricultural

373

Industrial System (CARS-049) and Guangdong Provincial Special Fund For Modern

374

Agriculture Industry Technology Innovation Teams, Department of Agriculture and

375

Rurual Affairs of Guangdong Province (2019KJ146). 15

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527 528

Figure legends

529

Fig. 1. Phylogenetic tree analysis of MyD88 genes. Genes in pink and yellow are

530

the two branches in bivalves. Pfm: P. f. martensii; Cgi: C. gigas; Mph: M. 22

531

philippinarum; Bpl: B. platifrons; Mye: M. yessoensis; Lgi: L. gigantean; Obi: O.

532

bimaculoides; Aca: A. californica; Has: H. sapiens; Dre: D. rerio.

533

Fig. 2. Gene structure of MyD88 genes.

534

Pfm: P. f. martensii; Cgi: C. gigas; Mph: M. philippinarum; Bpl: B. platifrons; Mye:

535

M. yessoensis; Lgi: L. gigantean; Obi: O. bimaculoides; Aca: A. californica; Has: H.

536

sapiens; Dre: D. rerio.

537

Fig. 3. Multi-alignment of MyD88 protein sequence.

538

The amino acids in purple indicate the conserved intron position; the amino acids in

539

yellow indicate the species–specific intron position. Numbers 0, 1, and 2 above the

540

intron position indicate intron insertion phases: 0, between two consecutive codons;

541

1, between the first and second codon positions; and 2, between the second and third

542

codon positions. Roman numerals I, II, III, IV, and V are the serial numbers named

543

by sequence.

544

Fig. 4. Nucleotide and amino acid sequence of PfmMyD88-2.

545

The 5ʹUTR and 3ʹUTR are indicated with small letters. The first read line showed the

546

DD domain, and the second read line showed the TIR domain. The black box

547

showed the initiation and stop codon.

548

Fig. 5. PfmMyD88-2 expression in tissues and after LPS stimulation.

549

a. Expression levels of PfmMyD88-2 genes in different tissues from P. f. martensii. b.

550

Expression patterns of PfmMyD88-2 genes at different times after LPS stimulation. A:

551

adductor muscle; F: foot; Go: gonad; He: hepatopancreas; M: mantle; H: hemocytes;

552

Gi: gill; The same letters above the bars represent no significant differences at the

553

p>0.05 level. Different letters above the bars represent significant differences at the

554

p<0.05 level. Error bars correspond to mean±SD.

555

Fig. 6. PfmMyD88-2 was involved in NF-κB signal pathway. 23

556

Relative luciferase activity of the luciferase reporter gene pNF-κB-Luc after

557

over-expression of PfmMyD88-2 in HEK293 cells. * p<0.05; error bars correspond to

558

mean±SD.

559

Fig. 7. PfmMyD88-2 was regulated by PfmmiR-4047.

560

a. Target interaction predicted by RNAhybrid; b. PfmmiR-4047 mimics significantly

561

downregulated the luciferase activity of the reporter Luc-PfmMyD88-2 detected by

562

dual

563

Luc-PfmMyD88-2 was the luciferase reporter plasmid containing the 3ʹUTR of the

564

PfmMyD88-2 genes. c. Expression of PfmmiR-4047 after injection of PfmmiR-4047

565

mimics. d. Expression of PfmMyD88-2 after over-expression of PfmmiR-4047.

566

Different letters above the bars represent significant differences at the p<0.05 level. *

567

p<0.05; error bars correspond to mean±SD.

568

Table 1. Primers used in this study.

569

Table 2. Characteristics of the MyD88 genes identified in this study.

570

Supplemental Figure 1. Expression of two MyD88 genes at different development

571

stages in C. gigas. E, egg; TC, two cells; FC, four cells; EM, early morula; M, morula;

572

B, blastula; RM, rotary movement; FS, free swimming; EG, early gastrula; G, gastrula;

573

T1, trochophore 1; T2, trochophore 2; T3, trochophore 3; T4, trochophore 4; T5,

574

trochophore 5; ED1, early D-larva 1; ED2, early D-larva 2; D1, D-larva 1; D2,

575

D-larva 2; D3, D-larva 3; D4, D-larva 4; D5, D-larva 5; D6, D-larva 6; D7, D-larva 7;

576

EU1, Early umbo larva 1; EU2, Early umbo larva 2; U1, umbo larva 1; U2, umbo

577

larva 2; U3, umbo larva 3; U4, umbo larva 4; U5, umbo larva 5; U6, umbo larva 6;

578

LU1, late umbo larva 1; LU2, late umbo larva 2; P1, pediveliger 1; P2, pediveliger 2;

579

S, spat; J, juvenile.

luciferase

analysis.

Control

was

24

the

empty vector

pMIR-REPORT.

Table 1. Primer sequence used in this study

Primer Name

Sequence(5’-3’)

PfmMyD88-2-5’RACE-outer

TGAAAGCCGATCAACTCTGCTAAACCATT

PfmMyD88-2-5’RACE-inner

AGGGCATCTAGTGAAATGTGCATAAAACGA

PfmMyD88-2-middle-F

GTGGGAAATAGACCAACAGCA

PfmMyD88-2-middle-R

ATCAGCAGTGAGAACTTGTGTTATT

PfmMyD88-2-3’RACE-outer

GAAAACGGGGTTTTTTGACAGACTTCGG

PfmMyD88-2-3’RACE-inner

TCGGGGGAAGAAAAATAACACAAGTTCT

M13-F

CGCCAGGGTTTTCCCAGTCACGAC

M13-R

AGCGGATAACAATTTCACACAGGA

Action

RACE

Colony PCR

PfmMyD88-2-qPCR-F

GATGGACTGGTTCTGGGACAC

PfmMyD88-2-qPCR-R

GTCCCAGAACCAGTCCATCA

PfmmiRNA-4047-F

CCAGACACTCAGAAACACGATT

PfmmiRNA-4047-R

TGCGTGTCGTGGAGTC (Common)

GAPDH-F

CACTCGCCAAGATAATCAACG

qRT-PCR

GAPDH-R

CCATTCCTGTCAACTTCCCAT

U6-F

ATTGGAACGATACAGAGAAGATT

U6-R

ATTTGCGTGTCATCCTTGC

PfmMyD88-2-reporter-F（SpeI）

CGGACTAGT+CACAAGTTCTCACTGCTGATCTATG

PfmMyD88-2-reporter-R（HindIII）

CCCAAGCTT+TTCTTTATTGAATCGTTTGATGTCA

PfmMyD88-2-3.1-F（SpeI）

CGCGGATCC+ATGACAAGTGCTCACGCTAGAAGTT

PfmMyD88-2-3.1-R（NotI）

ATTTGCGGCCGC+AACTTCTAGCGTGAGCACTTGTCAT

Reference gene (qRT-PCR)

Vector constructs

Species P. f. martensii C. gigas

M. yessoensis

M. philippinarum

B. platifrons A. californica O. bimaculoides L. gigantea H. sapiens D. rerio

Table 2. Characteristics of the MyD88 genes identified in this study. ID Length Locus Domain Pfm-AMQ81593.1 349 unknown DD-TIR Pfm-10008089(PfmMyD88-2) 473 scaffold1841：46877:54337:DD-TIR-3LCR Cgi-10007490 668 scaffold870:8942:14275:DD-TIR-3LCR Cgi-10013672 368 scaffold1714:1754:6575:DD-TIR Cgi-10026092 491 scaffold204:86156:93679:+ DD-TIR-2LCR Cgi-10026099 372 scaffold204:197004:201463:DD-TIR XP_021355224.1 472 NW_018403652.1:123052:128468+ DD-TIR-3LCR XP_021355236.1 443 NW_018403652.1:88782:101961+ DD-TIR-2LCR XP_021358176.1 371 NW_018407049.1：47537：57737DD-LCR-TIR-LCR XP_021358175.1 604 NW_018407049.1：83739：90667+ DD-LCR-TIR-3LCR XP_021354618.1 351 NW_018406224.1：1100646：1113660DD-TIR Mph_scaf_11876-0.10 450 scaf_11876：20944：44808+ DD-TIR-2LCR Mph_scaf_11876-0.8 430 scaf_11876：69193：89969+ DD-TIR-LCR Mph_scaf_11876-0.6 784 scaf_11876：125628：140296+ DD-LCR-TIR-2LCR Mph_scaf_7068-0.3 339 scaf_7068：31001：41961DD-TIR Bpl_scaf_24880-0.16 378 scaf_24880：142875：152446DD-TIR Bpl_scaf_26435-0.18 457 scaf_26435：35121：53370DD-TIR-2LCR XP_005094456.1 493 NW_004797351.1:799864:831987+ DD-TIR-3LCR XP_014784065.1 345 NW_014695501.1：51719：79016DD-TIR XP_009046476.1 389 NW_008707225.1:3323110:3327710+ DD-TIR-2LCR NP_001166038.2 312 NC_000003.12：38138478：38143022+ DD-TIR NP_997979.2 282 NC_007135.7：20188914：20192854DD-TIR

intron number unknown 7 4 5 6 5 5 5 5 5 5 5 5 5 4 5 5 9 5 5 4 4

Obi-XP 014784065.1 Mph-11876-0.8

99 24

Bpl-24880-0.16

24

Mye-XP 021354618.1 100

27

Mye-XP 021355224.1 Mye-XP 021355236.1 Pfm-10007755

98

81

Cgi-10026092 55

bivalve mollusc

Mph-11876-0.10 100

Bpl-26435-0.18 Cgi-10013672

100

Cgi-10026099

99

Pfm-AMQ81593.1

95

Mye-XP 021358176.1 27

58

Mph-7068-0.3 Mye-XP 021358175.1 Cgi-10007490

100 22

98

Mph scaf 11876-0.6

Lgi-XP 009046476.1 41

Aca-XP 005094456.1 Hsa-NP 001166038.2 100

0.10

Dre-NP 997979.2

Pfm-10008089 Cgi-10007490 Cgi-10013672 Cgi-10026092 Cgi-10026099 Mye-XP_021354618.1 Mye-XP_021355224.1 Mye-XP_021355236.1 Mye-XP_021358175.1 Mye-XP_021358176.1 Mph-scaf_11876-0.10 Mph-scaf_11876-0.6 Mph-scaf_11876-0.8 Mph-scaf_7068-0.3 Bpl-scaf_24880 Bpl-scaf_26435 Lgi-XP_009046476.1 Obi-XP_014784065.1 .Aca-XP_005094456.1 Dre-NP_997979.2 Hsa-NP_001166038.2

5' 0kb

Legend: CDS

3'

Intron

1kb

2kb

3kb

4kb

5kb

6kb

7kb

8kb

9kb

10kb

11kb

12kb

13kb

14kb

15kb

16kb

17kb

18kb

19kb

20kb

21kb

22kb

23kb

24kb

25kb

26kb

II（1）

I（1） 10 .

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D D D D D D D D D D D D D D D D D D D D D

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V L E C K T V IT V L E V IT V M E V M K V M K C L T V IS V IQ C L A IM L V IT V L L V IQ A L T V IT C L Q V L L V IS

E D E D E E D D E E D E E E E D E E D E E

Pfm-10008089 Cgi-10007490 Cgi-10013672 Cgi-10026092 Cgi-10026099 Mye-XP_021354618.1 Mye-XP_021355224.1 Mye-XP_021355236.1 Mye-XP_021358175.1 Mye-XP_021358176.1 Mph-11876-0.10 Mph_scaf_11876-0.6 Mph-11876-0.8 Mph-7068-0.3 Bpl-24880-0.16 Bpl-26435-0.18 Aca_XP_005094456.1 Lgi-XP_009046476.1 obi-XP_014784065.1 Hsa-NP_001166038.1 Dre-NP_997979.2

V L F L D V F F F F L F K K S I E I V D K

Pfm-10008089 Cgi-10007490 Cgi-10013672 Cgi-10026092 Cgi-10026099 Mye-XP_021354618.1 Mye-XP_021355224.1 Mye-XP_021355236.1 Mye-XP_021358175.1 Mye-XP_021358176.1 Mph-11876-0.10 Mph_scaf_11876-0.6 Mph-11876-0.8 Mph-7068-0.3 Bpl-24880-0.16 Bpl-26435-0.18 Aca_XP_005094456.1 Lgi-XP_009046476.1 Obi-XP_014784065.1 Hsa-NP_001166038.1 Dre-NP_997979.2

C Y C C IY C Y IY CW S Y S Y C C L C - IY C Y V Y C Y C Y C Y C Y C Y C Y C Y .

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Pfm-10008089 Cgi-10007490 Cgi-10013672 Cgi-10026092 Cgi-10026099 Mye-XP_021354618.1 Mye-XP_021355224.1 Mye-XP_021355236.1 Mye-XP_021358175.1 Mye-XP_021358176.1 Mph-11876-0.10 Mph_scaf_11876-0.6 Mph-11876-0.8 Mph-7068-0.3 Bpl-24880-0.16 Bpl-26435-0.18 Aca_XP_005094456.1 Lgi-XP_009046476.1 Obi-XP_014784065.1 Hsa-NP_001166038.1 Dre-NP_997979.2

V V I I V I V

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V L V L I L V L I L L L I L I L I L I L I L V L I L I L I L I L IM I L I L I L I L

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L A F L F L L L T P L S I V V L L M P V I

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D D D D D D D D D Q D E D D E D D D D D D

260

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G V M I D A T Y K - -M G IQ V K - -M -M D V V T A I V T A I G V S F E - -V S V P I D V F Y R L H L K - - I R L V I S V P I E - - I -C T I C T K Y K K E F K R P F

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V D V V V N N F K V V V V V E -

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F L F Y F F F Y H F Y A F F Y Y F Y F Y Y

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R L P Q P E V V C V Q L S P P Q Q K G P P

D K G D G D D D A G D K D H D D D D D C C

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LM F S L R L K L R M V L Q L Q S M IR LM Y G LM Q R LM LM L R L Q M H T K T Q

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DW L P DW DW DW DW EW EW T S HW EW H G EW EW EW EW DW EW EW SW VW

50 .

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F V IA F V F V F V F V F V F V L A F V F V D L F V F M F V F V F V F V F V F V F V

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Q T E E E K D D D E D S E D D E E M Q E E

M I L M L M M M I M M A C M C M L M M M M

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R L R R R R R R Q R R L R R R R R R R R R

L L L L L L L L V V L L L V L L L L L L L

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S K S A N A S K N A L K S S S S IA A A A K S S A C A A A E A K A Q V E A S A K A K

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A S V A V A A A S T S S S T S S S S A A A

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D H D P P E D H P E - I D H D H D D P P D H N C D H P Q D H D H P K D H D M P R P R

H I T D S N N V S D V I H V H V V T P D Y V S H Y V P IV Y I A I T V S V T A T Q

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L F F L F L L L L L L F L L L L L L L L L

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L V L G A L L V T A I L L V T L V L I IT V F L D F L - - R M L L L D R F L - - IM L L L L - - Q IM D V I G IT G V T

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P K P P P V P P K E P P P P P P P P P P P

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L Y L L L S L L P L L K L L L L L L F -

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D P R N IP K H N - - D P Q N N - - P DW E D P - D P - Q T K S R - - D P E R L V D G D D K E I - - E D - D P E H E M S D N A IE D F S D - - - - - - -

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F F C F C C F F F F C F C F C C F F C C C

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D Q D D D D D D H D D A D D D D N N D D D

D L D D D D D D L D D L C D C D D D V V V

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L P G L G G L P G L P G L P G L P G L P G L P G V G G L V G L P G L G G L IG L A G L IG L P G L A G L P G IP G L P G L P G

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S Y S A S A A A Y A S Y A E A S N A S C C

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IV（2）

190

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300 .

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V L A Y K M K L K M K L I L I L E L I L K L K K Q L V L Q L K L R F I L K L Q L Q L 280

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210

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C D C C C C C C D C C D C C C C C C C C C

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R R R R R L R R R R R R R R R R H R R R R

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IM IM I L IM I L V L I L I L L L IV V L I L V V V L V V V L V L I I I L V V V I

320

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100

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220 .

R R P R P E N N N K H K H K G H E H T D D

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N S K N K K L L N N N N H N R H Q K K Q D

S S S S S S S S S S S S S S S S S S S S S

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A P A A A V S S P P S P A E A S K P N K D

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A A A S A E A A A A A A A Y E A A A E E A

C C A A A C C C C A C C C C C C C C C C C

T 230

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D I D D D D D D S D D V D D D D D D D D D

F F F F F F F F F F F F F F F F F F F F F

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Q S Q Q Q M Q Q A Q Q A Q A R Q Q Q Q Q Q

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V K T Q L K V K L K T K C K S K T Q L K V K T Q V K L K L K V K T S T K IK T K T K

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F F F F F F F F F F F F F L F F F F F F F

A V A A A A A A A A A V A A A A A A A A A

H K H H H H H H K H H K H Q H H H F H L L

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A S C A C A A A T A A T S S S A A C S S S

L L L L L L L L L L L L L L L L L L L L L

120 . |

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- - -Q H - - - -S - - - L Y - - -Q F - - - L Y - - -Q Y - - -Q Y - - -Q F - - -Q M - - - IR - - - - S S V Q T - - -V T - - -E R - - -T I - - -T F - - - IC - - -Q L - - -T Y - - -E R - - -E T V（1）

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F Y Y F Y Y F F Y Y H Y F Y F Y Y Y F F

D V D D D D D D S V S D D D D D D D D D

A A A A A V A A A V I A A A A A A A A A

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S D S S S S S A D S A D E S N A N S S S C

P P P P P P P P P P P P P P P P P P P P P

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G D G G G A G G D G G D G G A G G G G G G

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A A A A A T A A A A A A A A A A A A S A A

R K R R R K R R R R R K R K R R R R R H R

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F C F F F F F F C L S F F F F F F F F F

V I V V V V V V V C V V V V I V V V I I 240

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.

S R S S S Q S N R Q S S K T R S E S Q Q T

K R K K K K K K R K K R K K N K K K K K K

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K K K K K K K K R R K K R R R K R K K R R

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L I V L V L L L I V L I L I I L I L L L L

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I I V I V V I I I I I I I I I I V V I I I

P P P P P P P P P P P P P P P P P P P P P

a

1 106 1 211 21 316 56 421 91 526 126 631 161 736 196 841 231 946 266 1051 301 1156 336 1261 1366 1471 1576

tgaacccggaagtttgtaaaacagaaacaagaataaggggaaattctatcaaagttcccccttgacaacataaggagaaggaagttgtgggaaatagaccaacag caaagaaacaaaatggccgacacgagtgtctaaatattctgttgtaacttttttacttgacttccaagtgtgtgtagatttgtcgatgtaatacccatgaacttt M T S A H A R S S L D S L V I V T E S G agacaagtttgacaagggaattttgtgtacaacatttcctgagcagaATGACAAGTGCTCACGCTAGAAGTTCTTTAGATTCTCTAGTGATTGTCACTGAATCTG G E C C L P D R F M H I S L D A L N H S A R R K I A L H M N L E S D V GAGGGGAATGCTGTTTACCTGATCGTTTTATGCACATTTCACTAGATGCCCTAAATCACAGTGCTCGTCGAAAAATTGCCTTGCATATGAACTTGGAATCTGATG P S D R N H L V S D Y N G L A E L I G F Q Y L E I K N F E R Q K S P T TACCAAGTGACAGAAATCACTTAGTAAGTGACTACAATGGTTTAGCAGAGTTGATCGGCTTTCAATACTTGGAAATCAAAAATTTTGAACGGCAGAAAAGTCCTA E E L L K E W C T R P D L E E P T L G K L W D F L V Q L G R V D V L E CAGAAGAACTGTTGAAGGAATGGTGCACACGTCCCGATCTCGAGGAACCTACGCTTGGAAAACTGTGGGATTTTCTTGTGCAACTCGGCCGTGTAGATGTATTAG E C Q Q M I I R D A E A Y L K M K E R M H N D Y A P L Q S N D V D S S AAGAATGTCAGCAGATGATAATTCGAGATGCCGAGGCTTATTTGAAGATGAAGGAGAGAATGCACAATGATTATGCACCTTTACAGAGCAATGACGTGGACAGTT S D G A M D H H I D E T L V L C R A D V T L G E P Q H F D A F V C Y N CATCAGACGGTGCTATGGATCATCATATAGATGAAACACTGGTCCTTTGTAGAGCTGATGTAACTCTTGGAGAGCCCCAGCACTTTGATGCCTTTGTCTGCTATA P E G E D L K F V K Q M I S V L E N E P H N L K L F V P W R D D L P G ACCCAGAAGGGGAAGATCTGAAGTTTGTCAAACAGATGATCAGTGTACTTGAAAACGAACCACATAATCTGAAACTGTTTGTGCCGTGGAGAGATGATCTGCCGG G S R Y V I D A K L I E S R C R R M V I I M S R N Y Q N S A A C D F Q GAGGTTCTCGATACGTTATAGATGCTAAACTCATAGAAAGCAGATGTCGTAGAATGGTGATTATAATGTCCAGGAATTACCAGAACAGTGCAGCTTGTGACTTTC V K F A H A L S P G A R S K K L I P V L I E P G V M I P Q V L R H V T AGGTTAAATTTGCCCATGCTCTCTCACCAGGTGCCAGGAGTAAGAAGCTGATTCCGGTTCTGATCGAGCCCGGCGTTATGATTCCTCAAGTGTTACGTCACGTGA L C D F T K R D L M D W F W D R L S K A I R A P L D P R N M T T F H K CGCTGTGTGATTTCACCAAGCGTGACTTGATGGACTGGTTCTGGGACAGACTGTCTAAAGCTATCAGGGCACCTCTAGACCCCAGAAACATGACAACTTTTCATA S S S S T S S S L S S S S W K K N N T S S H C AATCATCATCAAGTACATCATCTTCATTATCATCATCATCATGGAAGAAAAATAACACAAGTTCTCACTGCtgatctatgtgttttacttatgtacaattgtgct gaatgtgtaaaactatgtgtttgtgtgtagttcatatgtgtctgaataagtgtgtaagagagaggtttatttgtatttgttaaaacagctatgttttactatttc attacaaatacaaatatttctttttatgaattgtacaatgacagcttgttatatttgtgacatcaaacgattcaataaagaagaattattttgaaaaaaaaaaaa aaaaaaaaaaaaaaaa

b DD 0

LCR

TIR 100

200

300

a Relative expression

1.4

b

1.2

b

1 0.8

ab

0.6

ab

ab

0.4 0.2 0

b

a

A

a

F

Go

He

Relative expression

7

M

H

Gi

b

6

ab

5 4 3 2 1 0

ab a

a

ab

0h

2h

4h

a

8h

12 h

24 h

36 h

3 pcDNA3.1

Relative luciferase activiy

2.5

*

pcDNA3.1-PfmMyD88-2

2 1.5 1 0.5 0

pGL3-Basic

pNF-κB-Luc

a

b

PmMyD88-2 3’ AGTCTGTGTAT ACTTGATGTGTGTT

| | || | | |

|||

|||

5’ CCAGACACTCA-GAA--ACACGATT

PmmiR-4047

1.6

NC

1.4

PfmmiR-4047

1.2

0.14

b

0.12 0.1

0.08

0.06 0.04

a a

0.02 0

NC

DEPC

PfmmiR-4047

Relative expression

d

*

1 0.8 0.6 0.4 0.2 0

Relative expression

c

Relative luciferase activiy

mfe：-44.89 kcal/mol

1.8

0.12 0.1

Control a

Luc-PfmMyD88-2 a

0.08 0.06 0.04 0.02 0

b

NC

DEPC

PfmMyD88-2

1. A total of 18 MyD88 genes were identified from the mollusk bivalve. 2. MyD88 genes were expanded in bivalves. 3. Full sequence of PfmMyD88-2 was obtained. 4. PfmMyD88-2 was involved in the NF-kB pathway. 5. PfmMyD88-2 was regulated by PfmmiR-4047.