Expression, subcellular localization, and potential antiviral function of three interferon regulatory factors in the big-belly seahorse (Hippocampus abdominalis)

Journal Pre-proof Expression, subcellular localization, and potential antiviral function of three interferon regulatory factors in the big-belly seahorse (Hippocampus abdominalis) M.D. Neranjan Tharuka, Hyerim Yang, Jehee Lee PII:

S1050-4648(19)31069-1

DOI:

https://doi.org/10.1016/j.fsi.2019.11.026

Reference:

YFSIM 6595

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Fish and Shellfish Immunology

Received Date: 1 August 2019 Revised Date:

8 November 2019

Accepted Date: 13 November 2019

Please cite this article as: Tharuka MDN, Yang H, Lee J, Expression, subcellular localization, and potential antiviral function of three interferon regulatory factors in the big-belly seahorse (Hippocampus abdominalis), Fish and Shellfish Immunology (2020), doi: https://doi.org/10.1016/j.fsi.2019.11.026. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

1

Expression, subcellular localization, and potential antiviral function of three interferon

2

regulatory factors in the big-belly seahorse (Hippocampus abdominalis)

3 4

M.D. Neranjan Tharuka1,2, Hyerim Yang1,2 and Jehee Lee1,2*

5 6

1

7

Jeju Self-Governing Province 63243, Republic of Korea

8 9

2

Department of Marine Life Sciences & Fish Vaccine Research Center, Jeju National University,

Marine Science Institute, Jeju National University, Jeju Self-Governing Province 63333, Republic of Korea

10 11 12 13 14 15 16 17 18 19 20 21 22 23

*

Corresponding author Jehee Lee, Marine Molecular Genetics Lab, Department of Marine Life Sciences, College of Ocean Science, Jeju National University, 66 Jejudaehakno, Ara-Dong, Jeju, 690-756, Republic of Korea. Email: [email protected] (J. Lee)

24

Abstract

25

Interferon regulatory factors (IRFs) are among the most important transcription mediators and

26

have multiple biological functions, such as antiviral and antimicrobial defense, cell

27

differentiation, immune modulation, and apoptosis. Three IRF family members (HaIRF4-like,

28

HaIRF6, and HaIRF8) of the big belly seahorse (Hippocampus abdominalis) were molecularly

29

and functionally characterized at the sequence and transcriptional level. The coding sequences of

30

HaIRF4-like, HaIRF6, and HaIRF8 were 1214, 1485, and 1266 bp in length, encoding proteins

31

of size 46.21, 55.32, and 47.56 kDa, respectively. Potential viral transcription and replication

32

was detected against VHSV infection using qPCR in HaIRFs-transfected FHM cells. IRFs

33

significantly reduced viral gene expression at 24 h and 48 h post infection and the expression of

34

interferon-stimulated genes (ISGs) was modulated at transcriptional level upon HaIRF

35

overexpression in FHM cells. Subcellular HaIRF localization was observed using GFP-tagged

36

expression vectors in FHM cells. HaIRF4-like and HaIRF8 were localized to the nucleus,

37

whereas HaIRF6 was observed in the cytoplasm. All three IRFs were ubiquitously expressed in

38

all analyzed tissues of the big belly seahorse. The mRNA expression of IRF4-like, IRF6, and

39

IRF8 increased significantly post injection in the blood and gills following LPS, poly (I:C), and

40

Streptococcus iniae challenge. These findings demonstrate that seahorse IRFs are involved in

41

host defense mechanisms against immune stimulants and HaIRFs induce interferon and ISGs

42

which trigger antiviral activity against viral infections in the host.

43 44

Key words: Interferon regulatory factor; Big-belly seahorse; Immune challenge; mRNA

45

Expression pattern; Antiviral function.

46

1. Introduction

47

Interferon regulatory factors (IRFs) are transcription factors in interferon (IFN) signaling

48

pathways that play vital roles in immune responses against viral and bacterial invasions [1]. IRFs

49

positively or negatively regulate the expression of downstream IFN-related genes to trigger

50

cellular responses [2]. Eleven IRF (IRF1- IRF11) family members have currently been described

51

in vertebrates; although IRF10 is present in both Aves and Pisces species, IRF11 is found only in

52

Pisces species [3]. IRFs can be classified into four subfamilies on the basis of molecular

53

phylogenetics and their C-terminus: the IRF1 subfamily (IRF1, IRF2, and IRF11); the IRF3

54

subfamily (IRF3 and IRF7); the IRF4 subfamily (IRF4, IRF8, IRF9, and IRF10), and the IRF5

55

subfamily (IRF5 and IRF6) [4,5]. The C-terminus of all IRFs except IRF1 and IRF2 contains an

56

association domain known as the IRF-associated domain (IAD), which promotes precise

57

promotor targeting and regulates transcription by facilitating the formation of homodimers or

58

heterodimers with other transcription factors, including IRFs [6]. The C-terminus is the region

59

that determines the specific activity of each IRF, since the outer region of the IAD is less

60

conserved at the C-terminus [7]. At the N-terminus, all IRF proteins share five conserved

61

tryptophan residues within a DNA-binding domain (DBD) which form a helix-loop-helix motif.

62

The DBD binds to a consensus promoter sequence (A/GNGAAANNGAAACT) in target genes

63

known as the IFN stimulated response element (ISRE) [8,9].

64

The functions and regulation of IRF genes and their encoded proteins have been widely studied

65

in mammals [1,9,10]. All the homologues of IRF subfamilies have been identified in zebrafish

66

(Danio rerio) [11]; however, the IRF4 and IRF5 subfamilies are poorly described in teleost fish.

67

IRF4 and IRF8 are involved in the differentiation of myeloid progenitor cells and the

68

development of T cells and B cells [12,13]. Previous studies have demonstrated that IRF6 is

69

associated with the formation of connective tissues, yet the function of IRF6 has not yet been

70

elucidated in lower vertebrates [14]. Studies on teleost IRFs have mainly focused on their

71

expression pattern. The expression of IRF4 and IRF8 paralogues has been studied in rainbow

72

trout (Oncorhynchus mykiss), half-smooth tongue sole (Cynoglossus semilaevis), large yellow

73

croaker

74

lipopolysaccharide (LPS), polyinosinic:polycytidylic acid [poly (I:C)], and bacterial or viral

75

challenge [15–18]. In half-smooth tongue sole, IRF6 expression was studied following bacterial

76

and viral challenge [18]. Moreover, the overexpression of zebrafish IRF6 was shown to regulate

77

IFN expression and antiviral activity in vitro [19]. Characterizing IRFs and studying their

78

specific responses to pathogens may reveal the underlying immune mechanisms of teleost

79

species; however, the mRNA expression and antiviral activity of IRF4-like, IRF6, and IRF8 from

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the big-belly seahorse (Hippocampus abdominalis) (HaIRF4-like, HaIRF6, and HaIRF8) in

81

response to immunological stresses have not yet been studied.

82

The big-belly seahorse is a commercially important teleost that is used in the ornamental fish

83

industry, jewelry making, and traditional medicine in the Korean peninsula, China, and Japan

84

[20,21]. The immunity of the big-belly seahorse has become a concern since their natural

85

habitats and the mariculture industry have encountered various pathogenic infections, resulting in

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signs of seahorse extinction and a considerable loss of income to seahorse farming [22–24].

87

In this study, we functionally characterized the IRF4-like, IRF6 and IRF8 orthologs of the big-

88

belly seahorse and determined their mRNA expression profiles following immune challenge with

89

Streptococcus iniae, LPS, and poly (I:C). The coding sequences (CDS) of these IRFs were

90

cloned and we determined their subcellular localization, transcriptional induction of IFN-

(Larimichthys

crocea),

and

rockbream

(Oplegnathus

fasciatus)

following

91

stimulated genes (ISGs) and their antiviral capacities against viral hemorrhagic septicemia virus

92

(VHSV) in vitro.

93

2. Materials and Methods

94

2.1. Experimental seahorses and tissue isolation

95

Healthy seahorses (average body weight, 8 g) were purchased from the Korean marine fish

96

breeding center (Jeju Island, Republic of Korea) and acclimatized in aquarium tanks (300 L) for

97

one week prior to the experiments in the laboratory. The salinity (34 ± 0.6 g/L) and the

98

temperature (18 ± 2°C) of the aquarium water tanks were maintained. All fish experiments were

99

conducted according to the guidelines approved by the Animal Care and Use Committee of Jeju

100

National University. The liver, spleen, kidney, heart, gills, brain, skin, testis, ovary, intestine,

101

stomach, muscle, and pouch were obtained from six seahorses. Blood was drained by cutting the

102

verge of the tail and peripheral blood cells were obtained by centrifugation (3000 × g) at 4°C for

103

10 min. Tissues were immediately flash-frozen and stored at – 80°C.

104

2.2. Sequence identification and bioinformatics analysis of IRFs from the big-belly seahorse

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IRF4-like, IRF6, and IRF-8 were identified using the National Center for Biotechnology

106

Information

107

previously-generated transcriptome library of the big-belly seahorse [25]. The identified IRF

108

sequences were designated HaIRF4-like, HaIRF6, and HaIRF8, and all their open reading frames

109

(ORFs) were determined along with their complete amino acid sequences. The architecture of the

110

conserved domains, tertiary structures, and subcellular localization signals were predicted using

111

the NCBI conserved domain search (https://www.ncbi.nlm.nih.gov/cdd) with the ExPASy

112

prosite (http://prosite.expasy.org), SWISSMODEL (https://swissmodel.expasy.org/), and PSORT

(NCBI)

BLAST

program

(https://blast.ncbi.nlm.nih.gov/Blast.cgi)

with

a

113

II prediction (https://psort.hgc.jp/form2.html), respectively. Multiple sequence alignments and

114

pairwise sequence alignments of the amino acid sequences with different species were obtained

115

using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/) and EMBOSS needle

116

(https://www.ebi.ac.uk/Tools/psa/emboss_needle/), respectively. Nuclear localization sequences

117

and

118

mapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form.cgi)

119

(http://www.cbs.dtu.dk/services/NetPhos/). Physiochemical properties were estimated using the

120

ExPASy ProtParam tool (https://web.expasy.org/protparam/). Motif scan was used to identify

121

specific motifs in the domains (https://myhits.isb-sib.ch/cgi-bin/motif_scan). Phylogenetic trees

122

were built using MEGA version 6.0 with the neighbor-joining (nj) method and 10000 bootstrap

123

replicates.

124

2.3. Immunological challenge experiment

125

Seahorses with an average body weight of ~3 g were divided into four groups (three challenge

126

groups and one control group) of 30 seahorses each and were not fed for the duration of the

127

experiments. Injections of LPS (1.25 µg/µL), poly (I:C) (1.5 µg/µL), and S. iniae (105 CFU/µL)

128

were prepared with PBS (1×) solution at a final volume of 100 µL, with 100 µL of PBS (1×)

129

solution injected into the control group. All the injections were introduced intraperitoneally. The

130

gills and PBCs were isolated from five seahorses 0, 3, 6, 12, 24, 48, and 72 h post-injection (p.i.).

131

2.4. Total RNA purification and cDNA preparation

132

Total RNA was purified from the tissues of six healthy and five challenged seahorses at each

133

time point using RNAiso plus reagent (TaKaRa, Japan). The cleaning step was carried out by the

134

RNeasy spin column (Qiagen, Germany). The concentration and quality of the purified RNA

phosphorylation

sites

were

predicted

using and

cNLS

Mapper

NetPhos

3.1

(http://nlsServer

135

were determined using a spectrophotometer at 260 nm (µDrop Plate, Thermo Scientific, USA)

136

and agarose gel (1.5 %) electrophoresis, respectively. cDNA synthesis was performed in a

137

reaction volume of 20 µL with purified RNA (2.5 µg from each sample) using the

138

PrimerScriptTM II 1st strand cDNA Synthesis Kit (TaKaRa, Japan). Prepared cDNA was diluted

139

up to 40 fold in nuclease-free water and stored at – 80°C.

140

2.5. Transcriptional analysis of HaIRF4-like, HaIRF6, and HaIRF8.

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HaIRF4-like, HaIRF6, and HaIRF8 transcription was quantitatively analyzed in the

142

unchallenged and challenged samples using a Dice system III TP950 thermal cycler (TaKaRa,

143

Japan). Gene-specific primers for the HaIRFs and seahorse 40S ribosomal protein S7 (internal

144

control gene, ShRPS7; Accession no. KP780177) were designed according to the minimum

145

information for publication of quantitative real-time PCR experiments (MIQE) guidelines [26].

146

The quantitative PCR (qPCR) reaction mixture had a final volume of 10 µL, containing 3 µL

147

stored cDNA as a template, 1 µL of nuclease free water, 0.5 µL of each gene-specific primer (10

148

pmol/µL), and 5 µL of 2 × TaKaRa Ex TaqTM SYBR premix. The following qPCR thermal cycle

149

program was used: one cycle of 95 °C for 30 s, 45 amplification cycles of 95 °C for 5 s, 58 °C for

150

10 s, and 72 °C for 20 s, and a dissociation cycle of 95 °C for 15 s, 60 °C for 30 s, and 95 °C for

151

15 s. All qPCR reactions were performed in triplicate, and relative HaIRF mRNA expression was

152

calculated according to the Livak 2-∆∆Ct method [27], and the qPCR results were further

153

normalized to the corresponding PBS injected controls at each time point.

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2.6. Plasmid cloning

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Cloning primers were designed introducing appropriate restriction sites (Table 1). The coding

156

sequences of HaIRF4-like (EcoRI/XhoI), HaIRF6 (HindIII/XhoI), and HaIRF8 (HindIII/XhoI)

157

were cloned into their respective sites in pcDNA™3.1(+) expression vectors (Thermofisher,

158

USA). The same coding sequences of HaIRF4-like (XhoI/EcoRI), HaIRF6 (XhoI/HindIII), and

159

HaIRF8 (XhoI/HindIII) were cloned into the appropriate sites of the pEGFP-N1 vector (Clontech,

160

USA). The cloning of the inserted coding sequences was confirmed by sequence analysis

161

(Macrogen, Korea) and cloned plasmid constructions were isolated using a Plasmid Midi Kit

162

(Qiagen, Germany).

163

2.7. Cell line, virus, and transfection

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Fat Head Minnow (FHM) cells were cultured in L-15 medium containing 10% FBS, 100 U/mL

165

penicillin, and 100 µg/mL streptomycin in a 20ºC incubator. Cloned HaIRF4-like, HaIRF6, and

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HaIRF8-containing pcDNA 3.1(+) or empty pcDNA 3.1(+) vectors (1 µg) were transfected into

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FHM cells (105 cells/well) cultured in six-well plates using X-tremeGENETM 9 reagent (Sigma,

168

USA) according to the manufacturer’s instructions. VHSV titers were calculated as 50 % tissue

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culture infective dose (TCID50) according to the Reed-Muench method [28] from the previously

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prepared lab stock of a Korean isolate of VHSV (FWando05) [29]

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2.8. Subcellular HaIRF4-like, HaIRF6, and HaIRF8 localization

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To determine the subcellular localization of HaIRF4-like, HaIRF6, and HaIRF8 proteins, FHM

173

cells were transfected with pEGFP-N1/HaIRF4-like, pEGFP-N1/HaIRF6, pEGFP-N1/HaIRF8,

174

or an empty pEGFP-N1 vector and incubated for 24 h at 25°C until protein expression was

175

detected. DAPI (Invitrogen, USA) was used for nuclear staining according to the manufacturer’s

176

instructions. Briefly, cells were fixed with formaldehyde (4 %), washed twice with 1× PBS, then

177

DAPI was added, incubated at 37°C for 20 min, and washed with 1× PBS. Subcellular

178

localization was assessed using a fluorescence microscope (400×) (Leica Microsystems,

179

Germany) and Leica Application Suite X version 3.3 was used to processed the images.

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2.9. Detection of antiviral activity by gene expression and virus titer of VHSV

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FHM cells were transfected with HaIRF4-like, HaIRF6, and HaIRF8-containing or empty

182

pcDNA 3.1(+) vectors. After 24 h, the transfected FHM cells were infected with VHSV at 0.01 ×

183

the multiplicity of infection (MOI). Another series of transfected cells were not infected with the

184

virus. All samples were incubated for 24 and 48 h post infection, followed by RNA extraction,

185

cDNA synthesis, and qPCR analysis according to section 2.5. The elongation factor 1 alpha

186

(Accession No: AY643400) gene of the FHM cells was used as the internal control and VHSV

187

gene expression was detected using nucleocapsid protein transcripts (Accession No: AGS83377).

188

To detect virus replication, FHM cells were transfected and treated with VHSV at 0.01 × MOI as

189

mentioned above. The infected samples were incubated at 20°C for 72 h. Then, virus titers in

190

each sample were quantified as described by Reed and Muench method [28].

191 192

2.10. HaIRF4- like, HaIRF6, HaIRF8 overexpression

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FHM cells were transfected with HaIRF4-like, HaIRF6, and HaIRF8-containing or empty

194

pcDNA 3.1(+) vectors. All samples were incubated for 24 and 48 h, followed by RNA extraction,

195

cDNA synthesis, and qPCR analysis according to section 2.5. The elongation factor 1 alpha

196

(Accession No: AY643400) gene of the FHM cells was used as the internal control and qPCR

197

was carried out for interferon (Accession No: FN178457) and its downstream ISGs viperin

198

(Accession No: KM099177), Mx (Accession No: KM099175), TMEM173 (STING) (Accession

199

No: HE856620), and ubiquitin-like protein 1 (ISG15) (Accession No: KM099174) using gene

200

specific primers.

201

2.11. Statistical analysis

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All experiments were performed in triplicate and results are presented as the mean ±

203

standard deviation. The data were analyzed using Student’s t-tests to evaluate significant

204

differences between the groups. P < 0.05 was considered statistically significant. Virus

205

replication was compared using one-way analysis of variance (ANOVA) with Tukey’s multiple

206

comparison test.

207 208

3. Results

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3.1. Molecular characterization of HaIRF4-like, HaIRF6 and HaIRF8

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The complete open reading frames (ORFs) of HaIRF4-like (Accession No: MN046394), HaIRF6

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(Accession No: MN046395), and HaIRF8 (Accession No: MN046396) are 1214, 1485, and 1266

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bp in length, respectively. The deduced amino acid sequences of HaIRF4-like, HaIRF6, and

213

HaIRF8 were 404, 494, and 421 residues in length, with predicted molecular weights of 46.21,

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55.32, and 47.56 kDa, respectively. The theoretical isoelectric points (pI) were predicted as 8.65

215

(HaIRF4-like), 5.02 (HaIRF6), and 6.03 (HaIRF8). The subcellular localization signal prediction

216

results showed that HaIRF4-like and HaIRF8 were nuclear (73.9 % and 60.9 %) while HaIRF6

217

was mitochondrial (52.2%). NLS sequences of HaIRF4-like and HaIRF8 were predicted as

218

“DGVFIKRFCQGRVYWSGPLAPHTDRPNKLEREK”

219

“RQRHVTQKLLGHLERGLLLRANQEGIFIKRLCQS”, respectively. All HaIRFs contained an

220

N-terminal DBD with five tryptophan residues and a C-terminal IRF-associated domain (IAD)

and

221

(Fig. 1). IADs were located between the residues of 204 to 383 in HaIRF4-like, 249 to 432 in

222

HaIRF6, and 190 to 369 in HaIRF8, respectively (Fig.1). Predicted phosphorylation sites were

223

observed in the IAD domains of all HaIRFs. The HaIRFs and other vertebrate IRF orthologs

224

were aligned to compare sequence identities. HaIRF4-like was found to share the highest amino

225

residue identity (I) and similarity (S) with the olive flounder IRF4-like sequence (I, 58.9 %; S,

226

72.6 %), while HaIRF6 shared the highest identity and similarity with the Mandarin fish IRF6

227

sequence (I, 85.1 %; S, 91.3 %) and HaIRF8 shared the highest identity and similarity with the

228

Nile tilapia IRF8 sequence (I, 77.3 %; S, 86.8 %) (Fig. 1). To evaluate the evolutionary

229

relationship, individual phylogenetic trees were constructed. The individual tree was clearly

230

subdivided into respective subfamilies, with HaIRF4-like, HaIRF6, and HaIRF8 found in their

231

respective fish clusters (Fig. S1). A motif scan of HaIRF4-like, HaIRF6, and HaIRF8 revealed

232

the presence of serine-rich domains and protein kinase C phosphorylation sites in the C-terminal

233

region. Structural homology modelling was performed individually for the DBDs and IADs of

234

each HaIRF (Fig. S2). The DBD and IAD structures of HaIRF4-like were based on human IRF4

235

DBD (PDB: 2dll.1.A; I, 62.96 %) and IAD (PDB: 5bvi.1A; I, 42.86 %), respectively, the

236

HaIRF8 structure was modelled using human IRF4 DBD and IAD (I, 75.70 and 42.94 %,

237

respectively), and the HaIRF6 structure was modelled using human IRF4 DBD (PDB: 2dll.1.A; I,

238

46.17 %) and human IRF5 IAD (PDB: 3dsh.1; I, 60.17 %).

239

3.2. Subcellular localization

240

The subcellular localization of HaIRF4-like, HaIRF6, and HaIRF8 were determined by

241

transfecting pEGFP-N1/HaIRFs into FHM cells expressing seahorse IRF proteins tagged with

242

green fluorescence protein (GFP). DAPI staining indicated the nucleus in blue; HaIRF4-like/GFP

243

and HaIRF8/GFP expression were localized to the nucleus, whereas HaIRF6/GFP was mainly

244

observed in the cytoplasm and inside the nucleus of some cells (Fig. 2).

245

3.3. HaIRFs reduce viral gene transcription and virus titer in FHM cells

246

The possible effects of the HaIRFs on viral infection in FHM cells were observed after

247

transfection with pcDNA3.1+/HaIRFs or an empty pcDNA3.1+ vector. The viral load was

248

determined in the VHSV infected transfected cells at 24 and 48 h p.i. The FHM cells transfected

249

with pcDNA3.1+/HaIRF4-like, HaIRF6, and HaIRF8 exhibited significantly lower levels of

250

virus nucleocapsid transcripts than those transfected with the empty pcDNA3.1+ vector (control)

251

at 24 and 48 h p.i. (Fig. 3). Virus titer determination showed a significant decrease in TCID50/mL

252

values of HaIRF4-like, HaIRF6 and HaIRF8 transfected FHM cells (Fig. 4).

253

3.4. Effect of HaIRFs on downstream gene transcription

254

The effect of the HaIRFs on downstream gene transcription in FHM cells was observed after

255

their transfection with pcDNA3.1+/HaIRFs or an empty pcDNA3.1+ vector. Interferon (IFN)

256

and ISG transcription was determined in the overexpressed cells 24 and 48 h post transfection

257

(Fig. 5). HaIRF4-like overexpression upregulated the expression of IFN (2.1-fold), Mx (3.5-fold),

258

STING (~ 2.8-fold), and ISG15 (1.5-fold) at 24 h, as well STING (~ 3-fold) at 48 h. All four

259

ISGs and IFN were upregulated at 24 h following both HaIRF6 and HaIRF8 overexpression. Mx,

260

STING, and ISG15 were downregulated at 48 h following HaIRF6 expression, while HaIRF8

261

upregulated the expression of IFN (~ 2-fold), viperin (1.9-old), Mx (1.2-fold), and STING (4-

262

fold) at 48 h.

263

3.5. Quantitative detection of tissue-specific HaIRF mRNA expression patterns

264

Tissue-specific mRNA expression was determined using qPCR with gene-specific qPCR primers

265

under normal physiological conditions. Single product amplification was validated by obtaining

266

a single dissociation curve for the reference genes and HaIRFs. The highest tissue-specific

267

HaIRF4-like (Fig. 6A), HaIRF6 (Fig. 6B), and HaIRF8 (Fig. 6C) expression were observed in

268

the blood (186-fold), ovaries (28219.5-fold), and skin (163-fold) (Fig. 6), respectively, compared

269

to their lowest tissue expression (HaIRF4-like and HaIRF8, liver; HaIRF6, muscle).

270

3.5. Time-dependent transcription of HaIRFs upon immune challenge

271

In the blood, HaIRF4-like expression was significantly upregulated between 3 and 72 h p.i. upon

272

poly (I:C) challenge, peaking at 24 h.p.i (8-fold). LPS challenge only caused upregulation

273

between 6 and 24 h p.i, whereas S. iniae challenge resulted in significant upregulation at all time

274

points except for 3 h p.i (Fig. 6A). In the gills, HaIRF4-like expression peaked at 12 h following

275

all three challenges, with the highest expression levels observed following poly (I:C) challenge

276

(Fig. 6B). HaIRF6 transcription was significantly increased, peaking at 6 h p.i. in the blood and

277

gills upon LPS, poly (I:C), and S. iniae injection (Fig. 6C). In the gills, significant HaIRF6

278

expression was only observed at 6 h and 12 h p. i. following LPS challenge (Fig. 6D). Similarly,

279

in HaIRF8 expression peaked at 6 h p.i in the blood and gills following LPS, poly (I:C), and S.

280

iniae challenge (Fig. 7E, 7F) except at 72 h p.i. in the blood following S. iniae challenge.

281

4. Discussion

282

In vertebrates, IRFs have been identified as essential factors in the innate immune response to

283

viral and bacterial infections which trigger the IFN signaling pathway [9,30,31]. The role of fish

284

IRFs in immune defense mechanisms against viral and bacterial pathogens has not yet been

285

elucidated; hence, we characterized HaIRF4-like, HaIRF6, and HaIRF8 against immune

286

stimulants in vivo and assessed their antiviral activities in vitro. These HaIRFs possess putative

287

DBD domains with a pentad-tryptophan cluster similar to all other IRF family members in birds,

288

fish, and mammals [32,33]. Multiple alignments and sequence similarities of the HaIRFs with

289

vertebrate orthologous sequences confirmed the higher homology of the N-terminal DBD and C-

290

terminal IAD domains. Importantly, the C-terminal IAD domain in HaIRF4-like, HaIRF6, and

291

HaIRF8 is also involved in the regulation of cellular processes. Serine residues and

292

phosphorylation sites in these IADs are the target of virus-induced phosphorylation, allowing

293

them to interact with other IRFs to activate virus-inhibiting signaling pathways [34]. Tyrosine211

294

in the IAD domain of human IRF8 can be phosphorylated via TRAF6 activation, with IRF8

295

involved in gene transcription [35,36]. Phylogenetic analysis of vertebrate IRFs revealed that

296

seahorse IRFs clustered together with their corresponding fish orthologs, with distant

297

relationships to the mammalian IRFs. Additionally, 3D structural predictions of the HaIRFs were

298

generated based on the architecture of human IRF domains, since those are the only currently

299

available IRF crystallography data. Collectively, the in silico protein sequence analyses

300

confirmed the validity of the nomenclature of HaIRF4-like, HaIRF6, and HaIRF8 since they are

301

homologous to their fish and vertebrate IRF counterparts [3,16,17,37].

302

Zebrafish IRF4a is localized to the nucleus of epithelioma papulosum cyprinid (EPC) cells,

303

comparable to the subcellular localization of HaIRF4-like, and HaIRF8 observed in FHM cells

304

[38]. It was shown that IRF8 binds with the transcription factor Miz-1 in the nucleus of mouse

305

macrophages in order to provide an early innate immune response against intraphagosomal

306

pathogens such as Mycobacterium bovis and Salmonella enterica [13]. Moreover, a localization

307

study of crucian carp IRF9 confirmed the nuclear accumulation of IRF4 superfamily proteins

308

[39]. Furthermore, GFP fusion HaIRF6 was observed in the cytoplasm, as for zebrafish IRF6,

309

demonstrating the subcellular localization of teleost IRF6 [19]. It has also been found that TBK1

310

is localized in the cytoplasm [40] and that zebrafish TBK1 phosphorylates IRF6 [19]. It has been

311

clearly demonstrated that zebrafish IRF6 can induce IFN as a transcription factor; hence, the

312

distribution of HaIRF6 throughout the cytoplasm and nucleus may be obvious.

313

Vertebrate studies of the IRF family have reported their involvement in antiviral activities

314

[41,42]. AIV and NDV replication were restricted by overexpressed chicken IRF3 in chicken

315

embryonic fibroblast cells, while Chikungunya and Ross virus infection were highly detected in

316

the muscle tissues of irf1-/- mice [43]. In fish, the overexpression of sea perch IRF3 significantly

317

suppressed the gene expression of red spotted grouper nervous necrosis virus (RGNNV) in vitro

318

[44], whereas, zebrafish IRF1 and IRF6 reduced the transcription of SVCV viral genes in EPC

319

cells [19,45]. VHSV is recognized as one of the world’s major fish infectious diseases [46]. In

320

this study, we used VHSV to evaluate the antiviral activity of HaIRFs. Compared to the virus-

321

treated pcDNA3.1+ transfected controls, HaIRF-transfected-FHM cells exhibited significant

322

downregulation in VHSV nucleocapsid gene expression at 24 and 48 h post infection. Previous

323

fish antiviral studies have detected the viral content of VHSV using its nucleocapsid protein gene

324

expression [47–49]. Cells were treated with a MOD (0.01) of VHSV for this experiment since no

325

cytopathic effect was observed in the FHM cells until 48 h p.i. (data not shown). These results

326

suggest that HaIRF4-like, HaIRF6, and HaIRF8 overexpression is able to reduce viral

327

transcription in FHM cells in vitro. Both virus transcription and replication interfere with host

328

cellular functions [50]. Virus titer determination results also confirmed the attenuation of virus

329

replication in the presence of HaIRFs. It has been shown that the IAD domain contributes

330

towards antiviral properties and activates the double-stranded RNA activated factor 1 (DRAF1)

331

[51]. Therefore, these three HaIRFs with the IAD domain and other structural characteristics

332

mentioned above could trigger virus-inhibiting gene transcription pathways to exhibit potent

333

antiviral activities in overexpressed FHM cells. [34,52].

334

The effect of HaIRF4-like, HaIRF6, and HaIRF8 on downstream interferon and ISG expression

335

was analyzed 24 and 48 h post transfection. These results confirmed that all three HaIRFs have

336

the ability to trigger interferon expression [53]. Viperin Mx, and ISG-15 act on virus

337

transcription, replication, and protein translation in virus-infected cells [54–56]. STING responds

338

to elevated IFN by increasing its expression in order to detect foreign nucleic acids [57]. The

339

overexpression of HaIRF4-like upregulated viperin, Mx, and ISG-15 expression at 24 h and

340

STING at 48h; thus, HaIRF4-like may reduce viral transcription by inducing the expression of

341

ISGs [54]. Both HaIRF6 and HaIRF8 overexpression induced the expression of all four ISGs at

342

24 h and these were only upregulated at 48 h in HaIRF8-transfected cells. In the antiviral assay,

343

HaIRF8 reduced viral nucleoprotein transcription considerably more at 48 h than at 24 h,

344

suggesting that continuous ISG expression upon HaIRF8 overexpression could significantly limit

345

viral transcription compared to HaIRF4-like and HaIRF6.

346

Currently, the tissue-specific expression of HaIRFs has only been studied in a few fish species.

347

Tissue-specific expression was analyzed by qPCR and HaIRFs were constitutively detected in all

348

seahorse tissues at different levels. HaIRF4-like was strongly expressed in the blood and skin;

349

similarly, rockbream IRF4 (OfIRF4) levels were found to be higher in the blood and spleen [15].

350

Conversely, rainbow trout IRF4 and half-smooth tongue sole IRF4a and IRF4b showed highest

351

levels in the spleen and lowest levels in the liver and blood, respectively [16,18], and the highest

352

expression levels of large yellow croaker IRF4a and IRF4b and wuchang bream (Megalobrama

353

amblycephala) IRF4 were detected in the heart [17,58]. HaIRF8 was expressed in the skin and

354

kidney. Conversely, the tissue-specific expression of IRF8 in fish such as rainbow trout,

355

Japanese flounder, rockbream, and half-smooth tongue sole was highest in spleen, while in large

356

yellow croaker and wuchang bream the highest levels were in the heart, demonstrating that

357

tissue-specific expression patterns of IRF4 and IRF8 are somewhat similar between species

358

[15,16,18,33]. The tissues in which the lowest levels of HaIRF4-like and HaIRF8 expression

359

were detected were exactly compatible (liver, muscle, spleen, and brain, in ascending order). In

360

half-smooth tongue sole muscle and wuchang bream brain, IRF6 expression was highest in the

361

ovaries and gills, respectively, corresponding with strong HaIRF6 expression. However, these

362

HaIRFs were predominantly observed in tissues with abundant lymphomyeloid cell populations

363

such as the blood, gills, and kidneys [59,60]. Previous studies of tissue-specific fish IRF

364

expression have exhibited diverse spatial variations that may occur in a tissue- and species-

365

specific manner. Variation in IRF4, IRF6, and IRF8 expression have been noted in the kidney,

366

liver, and spleen of different fish species [15–18,33]. Blood contains macrophages and natural

367

killer cells that are primarily required for innate immunity. Fish gills are considered an important

368

mucosal tissue which assists in the initial prevention of pathogenic invasions [61]. Hence, we

369

used the blood and gills for the time-dependent challenge experiments with three immune

370

stimulants: poly (I:C) mimics viral dsRNA [62], LPS is a bacterial toxin in the outer membrane

371

of gram-negative bacteria [63], and gram-positive S. iniae is a severe aquaculture pathogen [64].

372

HaIRF4-like, HaIRF6, and HaIRF8 expression were detected by qPCR, demonstrating their

373

variation in the blood and gills after LPS, poly (I:C), and S. iniae challenge in seahorses.

374

Mammalian studies have confirmed the upregulation and functional importance of both IRF4 and

375

IRF8 in the differentiation of macrophages, dendritic (DC) cells, purified B/T cells, and

376

splenocytes [65–67]. Additionally, both IRFs are responsible for the regulation of pro-

377

inflammatory cytokines produced by macrophages and DC cells after LPS stimulation [68,69].

378

Although IRF4 expression is lower during the early stages of B cell development, it is

379

significantly upregulated during the maturation of B cells into plasma cells. IRF8 expression

380

maintains high levels during B cell development that oppose the IRF4 expression pattern and is

381

then downregulated during later stages [12,70]. Mammalian IRF8 regulates the signaling

382

between TLRs and IFN-γ; in particular, linking LPS-TLR4 and poly (I:C)-TLR3 to the IFN

383

system [68]. Similarly, HaIRF4-like was upregulated until 24 h p.i. in the blood and gills, yet

384

HaIRF8 was upregulated at all time points in the blood, but downregulated at 72 h p.i. in the gills

385

after LPS challenge. Rainbow trout LPS-stimulated splenocytes exhibited IRF4 downregulation

386

and no significant change in IRF8 expression [16], while OfIRF4 expression was also decreased

387

in both the kidneys and liver [15]. The HaIRF4-like transcriptional response to LPS was much

388

more similar to that of its mammalian IRF4 counterpart, suggesting that HaIRF4-like could

389

contribute and be regulated in a specific manner following LPS stimulation. Interestingly, in the

390

kidney and spleen of rockbream, IRF8 (OfIRF8) expression appears to be comparable with

391

HaIRF8 expression as well as mammalian IRF8 expression levels. Until now, IRF6 expression

392

had not been studied following LPS challenge in fish. A recent study in IRF6 knockout mice

393

confirmed that IRF6 is required for protection against LPS-triggered endotoxic shock [71].

394

Together with our results, this suggests that fish IRFs may possess conserved immune regulation

395

roles similar to mammals in response to LPS endotoxins.

396

The expression of all HaIRFs was upregulated immediately at 3 h and peaked before 24 h in the

397

blood and gills following poly (I:C) challenge, which is known to induce fish type I IFNs, IFN-γ,

398

antiviral IRFs, and ISGs [72–74]. Toll-like receptor 3 (TLR3) and TLR22 recognize fish

399

pathogen-associated molecular patterns (PAMPs) such as the poly (I:C), with TLR22 specific to

400

aquatic vertebrates [75]. IRF4-like expression was shown to be elevated in channel catfish

401

lymphoid cells by poly (I:C) [76], whereas in large yellow croaker and rockbream, poly (I:C)

402

induction caused IRF4 upregulation in both the spleen and liver, with no notable expression

403

changes in the kidney [15,17]. Poly (I:C) was shown to increase IRF8 expression in rainbow

404

trout, Japanese flounder, large yellow croaker, turbot, and rockbream immune tissues as well as

405

in non-immune tissues during the early phase post injection, showing that HaIRF8 expression is

406

similar to the expression of these fish species [15–17,33,77]. The main contrast between these

407

previous studies and this seahorse study is the observation of higher HaIRF4-like and HaIRF8

408

expression levels in the gills, which suggest that the seahorse has a better immune response

409

against viral invasion. In terms of poly (I:C) stimulation, fish IRF6 expression had not yet been

410

studied; however, our findings confirm that HaIRF4-like, HaIRF6, and HaIRF8 respond to and

411

protect the host from foreign viral stimuli.

412

IRF4 upregulation has been recorded when Helicobacter pylori infects the human gastric mucosa,

413

with mammalian IFR8 regulating innate immunity against pathogens such as Salmonella

414

typhimurium, Mycobacterium bovis, and M. tuberculosis by stimulating macrophages and

415

affecting antigen presentation in myeloid lineage cells [13,78,79]. Upon S. iniae infection,

416

significant HaIRF4-like and HaIRF8 expression were observed until 72 h p.i, except for

417

HaIRF4-like in the gill which was observed after 48 h p.i. Likewise, both IRF4a and IRF4b in

418

wuchang bream and IRF4b in Atlantic cod were elevated in the spleen and kidneys in response to

419

Aeromonas hydrophila and A. salmonicida, respectively [37,58]. In rockbream, OfIRF4 and

420

OfIRF8 were shown to be upregulated from 6 h p.i. after S. iniae infection [15]. Moreover, IRF8

421

mRNA expression was clearly upregulated in half-smooth tongue sole and large yellow croaker

422

after Edwardsiella tarda and Vibrio anguillarum challenge, respectively [17,18]. Upon A.

423

hydrophila challenge, wuchang bream IRF6 expression peaked within 12 h in the gills [58], with

424

early IRF6 expression observed in the spleen, head kidney, and liver of half smooth tongue sole

425

in response to V. harveyi [18]. Although fish IRF6 signaling pathways have been investigated

426

very little, significant HaIRF6 upregulation at earlier time points in the blood and gills upon all

427

three immune challenges confirmed the direct involvement of IRF6 in the host defense

428

mechanism. Together with the findings of previous fish studies, this study highlights the

429

importance of HaIRF4 variants, HaIRF6, and HaIRF8 in the antibacterial defense system of the

430

big belly seahorse.

431

Finally, we determined the complete CDS of HaIRF4-like, HaIRF6, and HaIRF8 and inserted the

432

CDS into the cloning vectors pcDNA3.1(+) and pEGFP-N1, separately. The constructed vectors

433

were then transfected into FHM cell lines and their antiviral properties against VHSV were

434

assessed. Nuclear localization confirmed that the individual HaIRFs could function as

435

transcription factors, while bioinformatics revealed that HaIRF4-like, HaIRF6, and HaIRF8 all

436

possess two main conserved domains, DBD and IAD. These domains exhibit higher similarity

437

with other vertebrate IRF4-like, HaIRF6, and IRF8 paralogues. The expression of all three

438

HaIRFs was detected in all tissues of healthy seahorses in a specific manner, with all genes

439

significantly upregulated in the blood and gill tissues soon after LPS, poly (I:C), and S. iniae

440

challenge. Based on the experimental results, we propose that HaIRF4-like, HaIRF6, and

441

HaIRF8 modulate the early innate immune defense mechanism against invading pathogenic

442

substances and may play a major role in antiviral activity in the big belly seahorse. Additional

443

research on teleost IRF systems could enhance our knowledge of fish immunology to overcome

444

current disease outbreaks in fish aquaculture.

445 446

Acknowledgments

447

This research was a part of a project titled ‘Fish Vaccine Research Center’, funded by the

448

Ministry of Oceans and Fisheries, Korea and supported by the National Research Foundation of

449

Korea (NRF) grant funded by the Korea government (MSIP) (NRF-2017R1C1B2008380).

450

Tables

451

Table 1. Nucleotide sequences of PCR and qPCR primers used in this study Primer name HaIRF4L_F HaIRF4L_R

452

HaIRF4L_F HaIRF4L_R

Application ORF amplification (for pcDNA3.1+) ORF amplification (for pEGEFP-N1)

HaIRF4L_qF HaIRF4L_qR

qPCR amplification

HaIRF6_F HaIRF6_R HaIRF6_F HaIRF6_R

ORF amplification (for pcDNA3.1+) ORF amplification (for pEGEFP-N1)

HaIRF6_qF HaIRF6_qR

qPCR amplification

HaIRF8_F HaIRF8_R HaIRF8_F HaIRF8_R

ORF amplification (for pcDNA3.1+) ORF amplification (for pEGEFP-N1)

HaIRF8_qF HaIRF8_qR

qPCR amplification

VHSV_qF VHSV_qR

qPCR amplification (nucleoprotein)

40S ribosomal protein S7_qF 40S ribosomal protein S7_qR

qPCR internal reference of seahorse

EF1α_qF EF1α_qR

qPCR internal reference of FHM cells

Sequence of primer (5’-3’) GAGAGAATTCGCTATGAAGATGCAGGAAGGGCCCAGGAT GC GAGAGACTCGAGTCACTGTGCTTCTTTCACTTGAC GAGAGACTCGAGATGAAGATGCAGGAAGGGCCCAGG GAGAGAATTCGCTGTGCTTCTTTCACTTGACTGGTGAC

GTCATGCACGGTGGACGCAATAA AGTTTGTTTGGCCTGTCAGTGTGG GAGAGAAGCTTGCTATGGCGGTGACGCCGCGACGTGTC GAGAGACTCGAGTCACTGGCCTTGCAGAGTGTGGAC GAGAGACTCGAGATGGCGGTGACGCCGCGACGTGTC GAGAGAAGCTTCTGGCCTTGCAGAGTGTGGACGAG TGGATCAGCTCCCTCCCAATGA TCCGAACAGCTCCTCTTGGTTGA GAGAAAGCTTGGCATGACGAATTCTGGAGGTCGAAGAC GAGAGACTCGAGTCAGGCGGTGATGGGCATGTTG GAGAGACTCGAGATGACGAATTCTGGAGGTCGAAGAC GAGAGAAAGCTTGGCGGTGATGGGCATGTTGTCCG GCATTATGGCCCTGTGCCTTGTAA ATCCGCTTATTCTTGGCCATGCTG TGTCTCAGATCAGTGGGAAGTACGC GGACCTCAGCGACAAGTTCGG

GCGGGAAGCATGTGGTCTTCATT ACTCCTGGGTCGCTTCTGCTTATT

GGCTGACTGTGCTGTGCTGAT GTGAAAGCCAGGAGGGCATGT

453 454

Figure Legends

455

Fig. 1. Multiple sequence alignment of (A) HaIRF4-like, (B) HaIRF6, and (C) HaIRF8 amino

456

acid sequences with their orthologous sequences from different organisms. Fully conserved,

457

strongly conserved, and weakly conserved amino acids are shown in black, dark gray, and light

458

gray, respectively. The N-terminal DNA binding domain is indicated by red lines and the IRF-

459

association domain (IAD) is indicated by blue lines. Conserved pentrad-tryptophan residues are

460

indicated in green boxes. NLS sequences of HaIRF4-like and HaIRF8 are indicated in red boxes.

461

Phosphorylation sites are indicated using blue stars in the IAD of all HaIRFs. At the end of each

462

sequence, the identity (I) and the similarity (S) of each ortholog with HaIRF4-like, HaIRF6, and

463

HaIRF8 are shown as percentages (%).

464

Fig. 2. Subcellular localization of (A) pEGFP-N1, (B) HaIRF4-like, (C) HaIRF6, and (D)

465

HaIRF8. FHM cells were transfected with an empty pEGFP-N1 vector or pEGFP-N1/HaIRFs.

466

GFP and GFP- tagged HaIRF (I) expression is indicated in green. The transfectants were stained

467

with DAPI (blue) indicating the nucleus (II). Merged localization results are shown in (III).

468

Fig. 3. In vitro effect of (A) HaIRF4-like, (B) HaIRF6, and (C) HaIRF8 on VHSV infection.

469

FHM cells were transfected with pcDNA3.1+/ HaIRFs or empty pcDNA3.1+ and infected with

470

VHSV 24 h post transfection. Viral nucleoprotein transcription was determined at 24 and 48 h p.i.

471

using qPCR. Results represent the mean ± SD of three replicates (n = 3). Statistically significant

472

values (P < 0.05) are indicated by an asterisk (*).

473

Fig. 4. Reduction of virus replication with HaIRFs. First and second columns represent the virus

474

titers obtained by un-transfected FHM cells and pcDNA3.1(+), respectively. Next columns

475

represent virus titers obtained by FHM cells transfected with HaIRF4-like, HaIRF6, and HaIRF8.

476

Results represent the mean ± SD of three replicates (n = 3) and analyzed using one-way ANOVA

477

with Tukey's comparison (p < 0.05). Statistical differences are indicated with lowercase letters.

478

Fig. 5. The effect of (A) HaIRF4-like, (B) HaIRF6, and (C) HaIRF8 overexpression on

479

interferon and downstream ISG transcription in FHM cells 24 h and 48 h post transfection. A1),

480

(B6), and (C11) graphs represent interferon expression (A2), (B7), and (C12) graphs represent

481

viperin expression. (A3), (B8), and (C13) graphs represent Mx expression. (A4), (B9), and (C14)

482

graphs represent STING expression. (A5), (B10), and (C15) graphs represent ISG15 expression.

483

Results represent the mean ± SD of three replicates (n = 3). Statistically significant values (P <

484

0.05) are indicated by an asterisk (*)

485

Fig. 6. Tissue-specific (A) HaIRF4-like, (B) HaIRF6 and (C) HaIRF8 mRNA expression profiles

486

(presented relative to the lowest expression of mRNA expression) in unchallenged H.

487

abdominalis. Results represent the mean ± SD of three replicates (n = 3).

488

Fig. 7. Relative expression analysis after injection with LPS, poly (I:C), and S. iniae by qPCR.

489

(A) HaIRF4-like (C) HaIRF6, and (E) HaIRF8 expression analysis in the blood. (B) HaIRF4-like

490

(D) HaIRF6, and (F) HaIRF8 expression analysis in the gills. Data present the mean ± SD of

491

three replicates (n = 3). Statistically significant values (P < 0.05) are indicated by an asterisk (*).

492

Fig. S1. Phylogenetic analysis of (A) HaIRF4-like, (B) HaIRF6, and (C) HaIRF8 with selected

493

full-length IRF4-like, IRF6, and IRF8 amino acid sequences from other species. The trees were

494

constructed using a neighbor-joining methods with 10000 replicates. Corresponding bootstrap

495

values for each protein are indicated on the branches and the NCBI accession numbers are

496

indicated along with the common names.

497

Fig. S2. Tertiary structures of HaIRF4-like (A, B), HaIRF6 (C, D), and HaIRF8 (E, F). The DNA

498

binding domains (DBD) are shown in (A), (C), and (E) and the IRF-association domains (IAD)

499

are shown in (B), (D), and (F). Strands, β-sheets, and α-helixes are represented using different

500

colors to highlight the contrasts between the domains. Conserved tryptophan (W) residues in the

501

DBD are shown in light purple along with their corresponding locations.

502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518

519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539

Fig. 1.

540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561

562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584

Fig. 2.

585 586

Fig. 3.

587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606

Fig. 4.

607 608

Fig. 5.

609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629

A.

630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657

B.

658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686

C.

687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708

Fig. 6.

709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730

Fig. 7.

731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753

754 755 756 757 758 759 760 761 762 763 764 765 766 767

Fig. S1.

768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789

790

791 792 793 794 795 796 797 798 799 800 801 802 803 804 805 806 807 808 809 810 811 812 813

Fig. S2.

814 815 816 817 818 819 820 821 822 823 824 825 826 827 828 829 830 831 832 833 834 835 836

837

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