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Influencing Mechanism of Ocean Acidification on Byssus Performance in the Pearl Oyster Pinctada fucata Shiguo Li, Chuang Liu, Aibin Zhan, Liping Xie, and Rongqing Zhang Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 12 Jun 2017 Downloaded from http://pubs.acs.org on June 12, 2017

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Environmental Science & Technology

1

Influencing Mechanism of Ocean Acidification on Byssus Performance in the Pearl

2

Oyster Pinctada fucata

3

Shiguo Li†‡§, Chuang Liu†∏§, Aibin Zhan‡, Liping Xie†∗, Rongqing Zhang†┴∗

4 5 6 7 8 9 10 11

†

Institute of Marine Biotechnology, School of Life Sciences, Tsinghua University, Beijing 100084,

China. ‡

Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085,

China. ∏

Tsinghua-Peking Joint Center for Life Sciences, School of Life Sciences, Tsinghua University,

Beijing 100084, China ┴

Department of Biotechnology and Biomedicine, Yangtze Delta Region Institute of Tsinghua

University, Jiaxing 314006, China

12 13

§

These authors contributed equally to this work and should be considered co-first authors

14 15

Corresponding Authors

16

Rongqing Zhang Ph.D.

17

Phone: +86 10 62772630. Fax: +86 10 62772899. E-mail: [email protected].

18

Liping Xie Ph.D.

19


20 21 22 23 24 25 26 27

ACS Paragon Plus Environment


28

ABSTRACT

29

The byssus is an important adhesive structure by which bivalves robustly adhere to underwater

30

substrates. It is susceptible to carbon dioxide-driven ocean acidification (OA). Previous investigations

31

have documented significant adverse effects of OA on the performance of byssal threads, but the

32

mechanisms remain largely unknown. In this study, multiple approaches were employed to reveal the

33

underlying mechanisms for the effects of OA on byssus production and mechanical properties in the

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pearl oyster Pinctada fucata. The results showed that OA altered the abundance and secondary

35

structure of byssal proteins and affected the contents of metal ions in distal threads, which together

36

reduced the byssus diameter and amplified byssus nanocavity, causing reductions in mechanical

37

properties (strength and extensibility). Expression analysis of key foot protein genes further confirmed

38

changes in byssal protein abundance. Moreover, comparative transcriptome analysis revealed

39

enrichment of ion transportation- and apoptosis-related categories, up-regulation of apoptosis-related

40

pathways and down-regulation of the “extracellular matrix-receptor interaction” pathway, which may

41

influence foot locomotion physiology, leading to a decrease in byssus production. This study provides

42

mechanistic insight into the effects of OA on pearl oyster byssus, which should broaden our overall

43

understanding of the impacts of OA on marine ecosystem.

44 45

INTRODUCTION

46

An increase in atmospheric carbon dioxide (CO2) concentration driven by human activities has led to a

47

decline in the surface seawater pH (termed ocean acidification, OA) since the onset of the Industrial

48

Revolution.1 It is proposed that the average pH value of the ocean surface will decrease by 0.3~0.4

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and 0.7~0.8 units by the end of 2100 and 2300 respectively, presenting a new set of challenges for

50

marine ecosystems.1-3 Mounting investigations show that OA has numerous adverse consequences to

51

the health and stability of marine ecosystems associated with the growth, reproduction and behavior of

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a wide variety of organisms.4 Marine calcifiers such as corals, sea urchins, oysters and mussels are

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highly susceptible to OA because they have special dissoluble calcium carbonate (CaCO3) structures.5-


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12

The bivalve byssus, a unique and valuable proteinaceous biomaterial in calcifiers, is also responsive

to OA.13-17

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Marine bivalves are ecologically dominant species in coastal ecosystems, partially because of their

57

capability to adhere strongly to different underwater substrates via the byssus, a holdfast structure that

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contains several byssal threads secreted within minutes by byssal glands in foot tissue. An individual

59

byssal thread is classified into three morphologically different regions: a crimped proximal portion, a

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stiff and smooth distal portion that is responsible for maintaining byssus extensibility and hardness,

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and an adhesive plaque facing various substrates.18 Constituent byssal proteins participating in

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byssogenesis and byssus bioadhesion are located in these three regions. Molecular characterization of

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byssal proteins and corresponding genes has been conducted in mussels,19,

64

scallops22. In mussels, more than ten proteins have been identified, including two thread matrix

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proteins (TMP and PTMP), six mussel foot proteins (MFP-1~6) and three prepolymerized collagens

66

(preCOLs): preCOL-C, preCOL-D and preCOL-NG.19 The histidine-metal crosslinkage-enriched

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preCOLs, distal thread-distributed MFP-1 and 3,4-dihydroxyphenylalanine (DOPA)-enriched MFP-

68

2~6 play key roles in byssus core framework formation, cuticle formation and plaque adhesion,

69

respectively.19, 23

20

pearl oysters21 and

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Changes in seawater temperature,24 salinity,25 current,26, hypoxia27 and metal ion concentration28

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have been shown to affect the strength and number of byssal threads in marine bivalves, with OA

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being a predominant factor.13-15, 17 Interactions between byssal proteins and metal ions contribute as

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load-bearing cross-links for surface adhesion.28 These chemical reactions are pH-dependent,29,

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thereby rendering the byssal threads easily affected by OA. For example, the percentage of attached

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byssal threads decreases with elevating CO2 concentration in the scallop Mimachlamys asperrima.16 In

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addition, OA reduces byssus strength and extensibility in the mussels Mytilus trossulus13 and M.

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coruscus17 and reduces byssus thickness in the pearl oyster Pinctada fucata.15 The byssus secretion

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rate, shed frequency, length and number are all decreased in the thick shell mussel M. coruscus

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incubated at lower pH, but not at higher temperatures.14, 17, 27 Overall, production of fewer, shorter and

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weaker byssuses negatively influences the growth, migration and predator-prey dynamics of marine


30


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bivalves, threatening the stability of coastal ecosystems and even the yield of shellfish aquaculture.

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Potential reasons for mussel byssus weakening are energy costs and adaptive escape14 and a recent

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study revealed that the molecular responses of byssal protein genes may contribute to weakened

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attachment of mussels exposed to OA.17 Despite such findings, the mechanisms by which OA

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influences byssus performance remain largely enigmatic. As byssogenesis and byssus attachment are

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not only chemical reactions but also biologically controlled processes,20-22 comprehensive elucidation

87

of the underlying mechanisms requires an in-depth and detailed understanding of two important

88

aspects: byssus material and foot physiology.

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The pearl oyster Pinctada fucata is an economically important bivalve for seawater pearl

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production worldwide. Compared with mussels, the byssus of pearl oysters has a greater economic

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implication due to its potential impacts on pearl quality during pearl sac development.26 The byssus

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color, number, morphology and protein component in pearl oysters are observably different from

93

those in mussels. In acute exposure experiments, OA reduced the byssus thickness but not the byssus

94

number in P. fucata.15 Our recent study uncovered the byssus ultrastructure and revealed the

95

involvements of Ca2+-stabilized nanocavities and byssal proteins, especially thrombospondin-1

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containing protein (TSP-1), in P. fucata byssus extensibility.21 Further analysis illustrated that Ca2+

97

can induce the self-assembly of adhesive proteins in this species, forming insoluble byssal threads

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underwater.29 Moreover, the foot transcriptome, byssal proteome21 and draft genome31 are available

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for P. fucata. These results suggest that P. fucata is an ideal species to study the bivalve byssus,

100

providing an excellent opportunity for resolving mechanical issues.

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Multiple approaches, including transcriptome sequencing, tandem mass spectrometry, Fourier

102

transform infrared spectroscopy, inductively coupled plasma mass spectrometry, transmission electron

103

microscopy and mechanical testing were utilized in this study. Our aim was to analyze global gene

104

expression profiles in foot tissue, detect protein structure and abundance, determine metal ion contents,

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observe ultrastructures, and test the mechanical properties of distal threads in P. fucata incubated

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under CO2-driven OA conditions to reveal the mechanism by which OA affects byssus production and

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mechanical properties. We focused on distal threads because they occupy approximately 80% of the


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total byssal length and are responsible for byssus extensibility and hardness. In P. fucata culture sea

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area, the pH of the surface seawater fluctuates from pH 8.1 in winter to pH 7.5 in summer. The mimicked

110

OA conditions cover the present and near-future oceanic pH variability, which may therefore be

111

helpful for accurately evaluating the effects of global climate change on coastal ecosystems and the

112

aquaculture industry due to the key ecological and economic niches of the pearl oysters.

113

MATERIALS AND METHODS

114

Pearl Oysters

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Two-year-old P. fucata individuals with similar shell lengths (6~7 cm) were sampled from the Pearl

116

Aquaculture Farm on the Leizhou Peninsula, China (20°24′ N, 110°0′E), in Feb 2016. Approximately

117

500 specimens were collected randomly by cutting the byssal threads off culture apparatuses without

118

damaging them. The temperature, salinity and pH of the seawater in these areas were 22.81 ± 0.73 °C,

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33.43 ± 0.56 psu and 8.14 ± 0.04, respectively. The pearl oysters were transported to the Institute of

120

Marine Biotechnology, Tsinghua University within 15 h of collection and maintained in a 500-L

121

aerated aquarium with artificial seawater (Formula Grade A Reef Sea Salt, Formula, Japan) for two

122

weeks at 23.42 ± 0.30 °C, pH 8.15 ± 0.05, and 33.36 ± 0.35 psu. The pearl oysters were fed mixed

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microalgae (50% Platymonas subcordiformis and 50% Isochrysis zhanjiangensis) at a concentration

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of 2.0 × 104 cells/mL twice daily.

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Experimental Design and Seawater Chemistry

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The CO2-driven OA conditions were manipulated using a seawater pH feedback system.10 Two pH

127

levels (pH 7.8 and pH 7.5) were designed to mimic the oceanic surface pH conditions predicted for the

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years 2100 and 2300; the ambient condition (pH 8.1) served as the control. After acclimation, existing

129

byssal threads of each individual were excised to the level of the byssus notch so that the newly

130

generated byssus could be fully exposed to OA condition. The pearl oysters were then separated into a

131

control group (pH 8.1) and two exposure groups (pH 7.8 and pH 7.5) and treated for 30 days. The

132

pearl oysters in control were reared and sampled in the same way as that in the treatment groups. The

133

pH value (NBS scale), temperature, total alkalinity (TA), and total dissolved inorganic carbon (DIC)

134

were monitored in each tank during exposure.10 To evaluate the stability of the OA system throughout



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the experiments, these parameters were subsequently used to calculate other parameters of carbonate

136

chemistry (partial pressure of carbon dioxide [pCO2], aragonite saturation state [Ωar], calcite

137

saturation state [Ωca], carbonate ion concentration [CO32-] and bicarbonate ion concentration [HCO3-])

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using the software CO2SYS32 with the dissociation constants K1, K2 and KSO4 (Supporting Information,

139

Table S1)33-35. Three biological replicates were designed using three independent tanks, and each tank

140

contained 50 individuals. The pearl oysters were fed the mixed microalgae at the rate described above.

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The byssal threads sampled from the same individuals were simultaneously used for multiple analyses

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because one pearl oyster can produce several distal threads.

143

Byssus Performance Test

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To assess the byssal thread performance, the number, diameter, strength (breaking force), and

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extensibility (breaking strain) were measured and tested following published methods.15,

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numbers and diameters of 90 individuals in three replicate tanks were recorded. One hundred and

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eighty distal threads from three replicate tanks with similar lengths were used to evaluate strength

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(force necessary to break the byssal thread) and extensibility (breaking strain at failure).

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Transmission Electron Microscopy

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To observe the byssal thread ultrastructure, ultrathin sections of distal threads were analyzed using

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transmission electron microscopy (TEM, Hitachi H-7650B, Japan), as described previously.21 In brief,

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30 distal threads from 30 individuals in each tank were fixed in 2.5% glutaraldehyde for 2 h followed

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by 1% osmic acid for 2 h, dehydrated in an ethanol series (50, 70, 80, 90, 100%), polymerized in Spurr

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resin at 60 °C for 48 h, sliced into sections with a diameter of 80 nm, and stained with 3% mixed

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uranyl acetate and lead citrate for 10 min. Micrographs of three replicates were directly obtained by

156

the microscope.

157

Inductively Coupled Plasma Mass Spectrometry

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To analyze the metal ion content in the byssal threads, distal threads were analyzed by inductively

159

coupled plasma mass spectrometry (ICP-MS, Agilent 7500c, New Castle, DE, USA) following a

160

previously described protocol.21 All the collected byssus was fully rinsed three times with Mili-Q

161

water to remove possible contaminants and then were dried in a closed container. Thirty distal threads


21, 36

The

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from 30 individuals in each tank were pooled together as a biological replicate. Three biological

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replicates were used to determine the contents of various metal ions, including calcium (Ca2+), silicon

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(Si4+), aluminum (Al3+), iron (Fe3+) and zinc (Zn2+), which are related to the mechanical properties of

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mussel byssal threads.24 Quality control (QC) of the metal ions analyses contained instrumental and

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run blanks, calibration tests and replicates. The contents of these metal ions are presented as mg

167

metalg byssus-1.

168

Thermal Gravimetric Analysis

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To analyze the thermal stability of byssal proteins, distal threads were examined by thermogravimetric

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analysis (TGA, 851e TG, Mettler, Schwarzenbach, Switzerland). Distal threads were collected using

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the method described above for ICP-MS and dried under a vacuum at 40 °C for 24 h. Five hundred

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milligrams of fragmented distal threads was heated from 40 to 800 °C at a heating rate of 10 °C/min in

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a nitrogen flow at a flow rate of 50 mL/min.

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Liquid Chromatography Coupled With Tandem Mass Spectrometry

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To explore the abundance of byssal proteins, liquid chromatography coupled with tandem mass

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spectrometry (LC-MS/MS) was employed using an LTQ Orbitrap Velos mass spectrometer with a

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Dionex U-3000 Rapid Separation nano LC system (Thermo Scientific, Waltham, USA) according to

178

published protocols.20 Ninety distal threads from 30 individuals in three replicate tanks were mixed

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together as a sample pool to perform sodium dodecyl sulfate polyacrylamide gel electrophoresis

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(SDS-PAGE). As shown in Figure S1, four bands were excised form the gel and used for LC-MS/MS

181

analyses.

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(http://www.uniprot.org/), the P. fucata draft genome database (http://marinegenomics.oist.jp/gallery/)

183

and identified byssal proteins in P. fucata byssus

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London, UK). For byssal proteins, the average values of the protein score, coverage and peptide

185

spectrum matches (PSMs) were calculated using data derived from the four bands. The abundances of

186

byssal proteins in the distal threads were compared between the control and OA group by assessing

187

changes in PSMs. The matched sequences with > 50% coverage were regarded as the most reliable

188

byssal proteins.

The

LC-MS/MS

results

were

searched

21

against

the

Swiss-Prot

database

using Mascot (2.1. version, Matrix Science,



189

Fourier Transform Infrared Spectroscopy

190

To detect the secondary structure of byssal proteins, Fourier transform infrared spectroscopy (FTIR)

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was employed using a spectrometer (VERTEX 70, Bruker Optics, Ettlingen, Germany), as previously

192

described.

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mixed together as a biological replicate. The distal threads were excised, rinsed with distilled water,

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air-dried and analyzed by FTIR. Briefly, FITR peaks at 1700-1580 cm–1 were decomposed by PeakFit

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4.12 (Systat Software Inc., Chicago, IL) with R2 >0.99. Baselines were corrected by linear background

196

subtraction with 3% tolerance. Local minima in the second derivative were used to identify peaks in

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the amide I region, which were fit as Gaussian curves. The peak position, height, and width were

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allowed to vary in a small range during an iterative least-squares method to minimize the residuals

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between reconstituted spectra and experimental spectra. In this case, the amine I region was

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deconvoluted to five curves, in which unknown region, β-sheet, unordered region, α-helix and β-turn

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structures were centered at wavenumbers of approximately 1612, 1628, 1643, 1660 and 1679 cm-1,

202

respectively.

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Transcriptome Sequencing and Real-time Quantitative PCR

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To analyze global gene expression profiles in foot tissue, foot transcriptomes were sequenced and

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compared. For each group, 90 foot tissues from 90 individuals in three replicate tanks were excised

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using sterilized surgical blades. The byssal threads were removed from the foot samples. These

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samples were then washed using sterilized seawater and mixed together as a sequencing sample. 22, 23

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RNA extraction, concentration determination, integrity analysis, cDNA library construction,

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transcriptome sequencing and sequence annotation were conducted using previously described

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protocols. 10 The foot transcriptome data have been deposited in the Sequence Read Archive (SRA) at

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the National Center for Biotechnology Information (NCBI) under accession number SRP097308. The

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false discovery rate (FDR) was used for statistical analysis, and genes with a FDR-adjusted p < 0.001

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and a fold change > 2.0 were regarded as differentially expressed unigenes (DGEs). For DEG analysis,

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Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations were

21

One hundred and eighty byssal threads from 90 individuals in three replicate tanks were


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performed using described previously methods.10 The FDR-adjusted p < 0.01 was used to identify the

216

most representative GO terms and KEGG pathways.

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To validate the transcriptome data, twenty genes containing 10 up-regulated and 10 down-regulated

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genes were selected randomly from the sequencing results, which cover a range of expression levels

219

of the DEGs. These genes were analyzed by real-time quantitative PCR (RT-qPCR) using a

220

StepOnePlus™ Real-Time PCR system (Applied Biosystems, Foster, CA, USA) with specific primers

221

(Table S2), as described previously.10, 37 Total RNA isolated from 90 pooled foot tissues of three

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biological replicates per treatment group was used to synthesize cDNA. The 18s rRNA gene10 of P.

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fucata was used as the reference gene, and relative fold changes were calculated following the 2−∆∆CT

224

method. 38

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To further explain changes in byssal protein abundance, the expression levels of TSP-1

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(Thrombospondin 1), P-UF1 (Foot uncharacterized protein 1), COL (Collagen alpha-4(VI) chain-like),

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and PER (Peroxidase) in foot tissue of P. fucata incubated under control and OA conditions were

228

analyzed by an RT-qPCR using the specific primers listed in Table S2.

229

Statistical Analysis

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The data presented are based on three biological replicates and expressed as the mean ± SD.

231

Differences between groups were evaluated using one-way (ANOVA), with the significance level set

232

at *p < 0.05 and **p < 0.01.

233 234

RESULTS

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Effects of OA on Byssus Performance

236

The number, diameter, breaking force and breaking strain of byssal threads were examined, all of

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which were significantly reduced with decreasing seawater pH (Figure 1). Compared with the control

238

(pH 8.1), the number of byssal threads was decreased by 41.59% and 63.55% in the pearl oysters

239

incubated at pH 7.8 and pH 7.5, respectively (Figure 1a, p < 0.01); the diameter decreased by 13.97%

240

at pH 7.8 and by 45.53% at pH 7.5 (Figure 1b, p < 0.01). The breaking force of distal threads was

241

0.71-fold at pH 7.8 and 0.42-fold at pH 7.5 (Figure 1c, p < 0.01), and the breaking strain of distal



242

threads was 0.87-fold at pH 7.8 and 0.72-fold at pH 7.5 less than that in the control at pH 8.1 (Figure

243

1d, p < 0.01). These results demonstrate that distal thread performance was negatively influenced by

244

OA.

245

Effects of OA on Byssus Ultrastructure

246

TEM images showed that normal byssal threads are composed of abundant compact protein fibers

247

with a size of 80~200 nm and a few nanocavities among the protein fibers with a size of

248

approximately 0.025 µm2 (Figure 2a). After exposure to pH 7.5 for 30 days, the shape and size did not

249

change, but the number of protein fibers appeared to be less than that in the control at pH 8.1. The size

250

of each nanocavity in distal threads incubated at pH 7.5 was approximately 40-fold higher

251

(approximately 1 µm2) than that in the control at pH 8.1 (Figure 2b). These results indicate that OA

252

significantly increased the nanocavity size, thus altering the distal thread ultrastructure.

253

Effects of OA on Byssus Metal Ion Content

254

The contents of five metal ions in distal threads under different pH conditions were determined by

255

ICP-MS. As shown in Table 1, the concentration of each metal ion (Ca2+, Si4+ and Al3+) was

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significantly decreased in distal threads exposed to pH 7.5 compared to the control; an exception was

257

for Fe3+ and Zn2+, with higher contents at pH 7.5. Notably, the Ca2+ content was decreased by 99.63%

258

in distal threads exposed to pH 7.5 compared with the control. These results show that OA

259

significantly influenced the metal ion contents of distal threads.

260

Effects of OA on Thermal Stability

261

With increasing temperature from 40 to 800 °C, three trends in distal thread weight loss were observed

262

in the control (Figure 3a). The first rapid weight loss began at 90 °C, which was caused by a loss of

263

moisture. For the second and third trends, the rate of weight loss became significant and rapid when

264

the temperature was higher than 318 °C and 584 °C, respectively. These two rapid decreases were

265

induced by degradation of the organic matrix, possibly byssal proteins. It can be seen that the

266

derivative weight curve at pH 8.1 was gradual (Figure 3a), and the derivative weight curve at pH 7.5

267

was made of some stairs (Figure 3b). At 400 °C, 450 °C, 500 °C and 550 °C, the remaining weights of

268

byssus samples were ~52%, ~47%, ~42% , and ~30% respectively for pH 8.1 (Figure 3a), while ~52%,


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~42%, ~32% and ~18% respectively for pH 7.5 (Figure 3b). These data suggest that byssus at pH 7.5

270

condition were more susceptible to high temperature and the thermal stability of byssal proteins was

271

altered by the OA treatment.

272

Effects of OA on Byssal Protein Abundance

273

Based on LC-MS/MS data, byssal proteins were identified throughout searches against Swiss-Prot and

274

the draft genome database, and the abundances of these proteins were further analyzed (Table 2 and

275

Table S3). TSP-1, P-UF1 and COL are predicted to be structural byssus proteins, and PER may be

276

involved in oxidative reactions underwater. In the control, the coverage of the four byssal proteins

277

TSP-1, P-UF1, COL and PER was more than 50%, and the corresponding PSMs were 268.00 ± 17.88,

278

303.00 ± 58.93, 203.77 ± 13.79 and 74.25 ± 10.84, respectively. For the OA group, the coverage of six

279

byssal proteins, TSP-1, P-UF1, COL, PER, actin and tubulin, was above 50%; the PSMs for TSP-1

280

(146.25 ± 18.21), COL (24.16 ± 2.10), P-UF1 (137.75 ± 15.46) and PER (144.25 ± 32.18) were 0.55-

281

fold, 0.12-fold, 0.45-fold and 1.94-fold (p < 0.01) the control values. All of these results suggest that

282

byssal protein abundance in distal threads was significantly influenced by OA.

283

Effects of OA on Byssal Protein Secondary Structure

284

Quantitative determination of structural changes in byssal proteins in distal threads was conducted

285

using FTIR at the amide I regions (Figure 4). FTIR has been used for determining the secondary

286

structures of protein fibers such as silk and mussel byssus. Compared with the control (pH 8.1), the

287

peak areas for the five structures of byssal proteins were altered by OA (pH 7.5). According to curve-

288

fitting analyses, the control byssal proteins contain 20% α-helix, 27% β-sheet, 12% β-turn, 27%

289

unordered structure and 14% unknown structure (Figure 4a). After exposure to OA, the β-sheet

290

content was decreased from 27% to 17%, and the unknown structure content decreased from 14% to

291

8%. The α-helix and the β-turn contents were increased to 27% and 21%, respectively, whereas no

292

significant change was observed for the unordered structure (Figure 4b). These results indicate that

293

OA significantly altered secondary structure of byssal proteins.

294

Effects of OA on the Foot Gene Expression Profile



295

Using transcriptome sequencing, 53,562,750 clean reads with a Q20 of 98.53 and 52,616,814 clean

296

reads with a Q20 of 97.26 were obtained from foot tissue of P. fucata incubated in control (pH 8.1)

297

and acidified seawater (pH 7.5), respectively (Table S4). The assembly based on the contigs yielded

298

98,710 unigenes with an N50 of 1908 bp and an average length of 817 bp in the control and 106,462

299

unigenes with an N50 of 2051 bp and an average length of 867 bp in the OA group (Table S5),

300

showing that de novo sequencing and assembly were able to generate high quality sequences.

301

Based on the cutoff of p < 0.001 and fold change > 2, 19,810 DEGs were obtained for foot tissue

302

from the OA group compared with the control, including 14,027 up-regulated unigenes and 5783

303

down-regulated unigenes (Figure 5a, Table S6). The fold changes for these DGEs were mainly

304

distributed from 21~24-fold and 2-1~2-4-fold, accounting for 74.02% and 67.15% of the total,

305

respectively (Figure 5b). Linear regression was observed for the fold changes calculated from RT-

306

qPCR and transcriptome data (Figure S2, R2 = 0.91), validating the accuracy and reliability of the

307

transcriptome data.

308

Based on RT-qPCR results, the expression levels of TSP-1, P-UF1 and COL were down-regulated

309

by 5.46-fold, 12.73-fold and 17.03-fold and that of PER up-regulated by 4.29-fold in the OA group

310

(pH 7.5) compared with the control (pH 8.1). These expression trends were in agreement with those

311

revealed by transcriptome analysis (Table S7). The observed changes in expression of key functional

312

genes in foot tissue were also consistent with the changes in abundance of byssal proteins detected by

313

LC-MS/MS.

314

GO enrichment analysis of DGEs indicated that a “proton-transporting two-sector ATPase complex”

315

in “Cellular Component” and “autophagic cell death” in “Biological process” were significantly

316

influenced by OA (pH 7.5) in foot tissue (Table 3). KEGG enrichment analyses revealed only one

317

pathway, “ECM-receptor interaction” (Figure S3), to be significantly down-regulated, whereas several

318

pathways, including “lysosome” (Figure S4), “ubiquitin mediated proteolysis” (Figure S5), apoptosis

319

(Figure S6), “NF-kappa β signaling pathway” (Figure S7) and “glycosaminoglycan degradation”

320

(Figure S8), were significantly up-regulated in foot tissue under the OA condition (Table 4). These

321

results show that foot gene expression profiles were significantly altered by OA.


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322 323

DISSCUSSION

324

Marine bivalves attach to various underwater substrata via their robust byssus, maintaining the

325

stability of marine ecosystems as well as commercial yields. Therefore, exploration of the factors

326

influencing byssus performance has great scientific and economic significance. This study

327

investigated the effects of OA on byssus performance in the pearl oyster P. fucata and revealed the

328

underlying mechanism at physiological, biochemical and molecular levels.

329

Our findings illustrate that OA reduces the byssus number and weakens the mechanical properties

330

(strength and extensibility) of distal threads in P. fucata, consistent with the results for the mussels M.

331

coruscus14, 17 and M. trossulus13 incubated at similar pH levels. These findings demonstrate that the

332

effect of OA on the byssus in marine bivalves is a general phenomenon. Under OA, the energy budget

333

of marine organisms will be reassigned, and to maintain survival, more metabolic energy may be

334

allocated to critical physiological processes such as ion and acid-base homeostasis.39 It has become

335

clear that this energy trade-off will negatively influence several other physiological aspects of marine

336

bivalves, including calcification and byssus formation.17, 40 In this regard, pearl oysters may adopt a

337

trade-off strategy, yielding an obvious decrease in byssus production.

338

As mentioned above, the negative effects of OA on the performance of distal threads increase with

339

a greater degree of acidification (pH 7.8 and pH 7.5); thus, the pearl oysters exposed to pH 7.5

340

seawater in this study were regarded as acidified samples for analyzing the influencing mechanism.

341

The results confirm that OA results in byssus weakening by altering the ultrastructure and morphology

342

of byssal threads. The distal thread of P. fucata comprises a compact cuticle and a core filled with

343

nanoscale protein fibers. Between the fibers are massive nanocavities, which are inferred to be

344

responsible for the extensibility of the threads.21 Compared with the control, the nanocavity area

345

increased by almost 40-fold in distal threads exposed to OA, and the diameter decreased by nearly

346

one-fold. These structural and morphological changes undoubtedly result in significant declines in the

347

mechanical properties of the distal threads in pearl oysters.



348

The structural integrity of byssal threads in marine bivalves is chiefly controlled by the balance of

349

metal ions and byssal proteins. The equilibrium of metal ions and byssus structure may reflect

350

seasonal changes in byssus mechanical properties of the blue mussel M. edulis.24 It was also reported

351

that expanded nanocavities are the result of a disturbed fiber structure in the distal threads of P. fucata,

352

which was substantially attributed to Ca2+ deficiency,21 meaning that metal ions are essential for the

353

distal thread ultrastructure. This result is in accordance with Harrington and Waite (2007),41 Holten-

354

Andersen et al. (2009)42 and Seguin-Heine et al (2014),24 who investigated changes in the strength of

355

byssal threads in the mussels M. edulis, M. californianus and M. galloprovincialis as a function of the

356

concentrations of metal ions, such as Mo2+, Zn2+, Cu2+, Fe3+, Al3+, and especially Ca2+.

357

Indeed, metal ions are involved in the crosslinking of byssal proteins through interactions with

358

amino acids such as histidine, cysteine and DOPA. Increasing evidence supports the key role of the

359

interaction between metal ions and amino acids in regulating the mechanical responses of distal

360

threads.

361

self-assembly of these proteins from the nanoscale to microscale.43 Consequently, the observed

362

significant changes in the contents of Ca2+, Si4+, Zn2+, Fe3+ and Al3+ in the distal threads of pearl

363

oysters disturbed the metal ion equilibriums, which may amplify the size of metal-stabilized

364

nanocavities, leading to a decline in distal thread mechanical properties. Investigation on the mussel M.

365

galloprovincialis also showed that declines in Zn2+ and Fe3+ contents in byssal threads had a negative

366

impact on mechanical properties, which could be attributed to the integration of these metal ions from

367

seawater into the byssus structure.24 Accordingly, we infer that pearl oyster byssus exposed to OA can

368

accumulate Fe3+ and Zn2+ to compensate for the loss of Ca2+, Al3+ and Si4+, leading to increase in the

369

contents of Fe3+ and Zn2+.

29, 30

Importantly, Ca2+ can interact with specific domains of byssal proteins and mediate the

370

In addition to metal ions, the abundance and secondary structure of byssal proteins were altered in

371

the distal threads of P. fucata reared under the OA condition, which may also be drivers of changes in

372

the ultrastructure, morphology and subsequent mechanical properties. Eighteen and nine abundant

373

byssal proteins have been isolated from the distal threads of the mussel M. coruscus20 and the scallop

374

C. farreri,22 respectively. Inconsistent with these findings, our earlier study isolated 14 proteins from


Page 14 of 34

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375

P. fucata, including low-complexity repetitive domain-enriched proteins, enzymes and cytoskeleton-

376

related proteins.21 TSP-1, the first identified byssal protein from P. fucata, has been proven to be

377

associated with the byssus extensibility. On the one hand, the abundance of byssal proteins TSP-1, P-

378

UF1, COL and PER was significantly influenced by OA in this study, which would cause changes in

379

byssal fiber shapes and an increase in nanocavity areas. On the other hand, the contents of unknown

380

structures and β-sheets in byssal proteins declined under OA stress, which would also induce changes

381

in byssus ultrastructure. Under the acidic condition (pH≈4.0) histidine-rich preCOL precursors in

382

storage vesicles of mussel foot tissue are soluble and exhibit a mixture of various secondary structures;

383

under the control condition (pH≈8.0), highly concentrated preCOLs self-assemble into a compact

384

phase of a chain-like β-sheet structure.

385

secondary structure transitions of byssal proteins. Similar to the present study, decreases in the

386

contents of the unknown structure and β-sheets disrupted the regular ultrastructure and mechanical

387

properties of byssal threads in the pearl oyster21 and silks in the caddis fly44, suggesting the

388

importance of protein secondary structure for the structures and performance of these biomaterials.

44

These findings confirm that pH changes can trigger

389

Notably, the mussel M. trossulus showed positive growth with a decrease in seawater pH, and

390

physiological metrics were also normal, suggesting that the effects of OA on byssus are not the result

391

of deteriorating body physiology.13 Our sequencing data support this conclusion and demonstrate that

392

changes in foot physiology may give rise to reduced byssus performance. Comparing the global

393

transcriptome of foot tissue from P. fucata treated with OA to that of the control provides an

394

opportunity to examine the gene-function relationship among gene expressions, foot physiology and

395

byssus performance. GO analysis showed that the “Proton-transporting two-sector ATPase complex”

396

category in the “Cellular component” term was down-regulated in the foot tissue exposed to OA.

397

Proton-transporting two-sector ATPase is a large protein complex that catalyzes the synthesis or

398

hydrolysis of ATP via a rotational mechanism, coupled to the transport of protons across a membrane.

399

It is well known that ion and acid-base regulation is an effective strategy implemented by the mantle

400

of marine bivalves to compensate for OA-induced imbalance in intracellular and extracellular ion

401

concentrations.45 Similar to the mantle, our GO results revealed the possible existence of an ion and



402

acid-base regulatory mechanism in the foot tissue of pearl oysters in OA acclimation. The down-

403

regulation of “Proton-transporting two-sector ATPase” may cause the changes in acid-base physiology

404

of foot tissue to acclimate to OA condition, resulting in the decline of byssus production. Furthermore,

405

up-regulation of “Autophagic cell death” was observed in the “Biological process” term, indicating

406

that the foot tissue might have been injured by OA, invoking the cell death process. This conclusion is

407

in accordance with the KEGG analysis results, whereby up-regulated “Apoptosis”, “Lysosome” and

408

“Ubiquitin mediated proteolysis” pathways were significantly enriched. “Lysosome” and “Ubiquitin

409

mediated proteolysis” are crucial pathways during autophagic cell death and apoptosis, by which

410

cellular debris and dead organelles of marine organisms under the stresses of climate change factors

411

are removed and several biological molecules, including carbohydrates, proteins, nucleic acids and

412

lipids, are degreaded.46, 47 In “Apoptosis” pathway, CASPs are the members of a large gene family

413

encoding cysteine proteases, which play crucial roles in apoptosis. Caspase activity significantly

414

increased in the corals Pocillopora damicornis and Oculina patagonica exposed to OA for 15~30

415

days.48 Study on the oyster C. gigas showed that Caspase-3 and Caspase-9 were both activated by OA

416

exposure.49 These investigations suggest closely linkage between increased cell apoptosis and OA.

417

After exposure to OA condition, the expression levels of CASP3, CASP6, CASP7, CASP8, CASP9 and

418

CASP10 were all increased, indicating that “Apoptosis” pathway may be activated in the pearl oyster

419

P. fucata. Our results therefore support the above conclusion and confirm that OA could induce cell

420

apoptosis in foot tissue. The “NF-kappa β signaling pathway” regulates apoptosis in mammals.50 In

421

the mussel P. grandis, the responses of pro-apoptotic pathways to climate change were directly

422

mediated through NF-kappa β activation.51 Accordingly, by transmitting the stress signal to these

423

specific functional pathways, up-regulation of the NF-kappa βpathway may be involved in the

424

responses of the foot to OA.

425

Transcriptome analysis shows that OA affected byssus performance by reducing the metabolic level

426

of the “ECM-receptor interaction” pathway. The extracellular matrix (ECM) consists of a complex

427

mixture of structural and functional macromolecules, including collagen, tenascin and laminin,52

428

which are essential for tissue architecture and play critical roles in the control of migration, adhesion,


Page 16 of 34

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429

differentiation and proliferation. The down-regulated “ECM-receptor interaction” pathway reflects

430

lower expressions of genes encoding these macromolecules in the pearl oysters, which could influence

431

foot locomotion. The foot locomotion changes will reduce byssus production because foot

432

extensibility is a prerequisite for the formation of byssal threads.53,54 Moreover, among these

433

macromolecules, preCOLs are important components of byssal proteins and participate in maintaining

434

the rigidity and strength of byssal threads. Down-regulation of preCOL-D, a collagen gene encoding

435

preCOL-D (required for distal threads), has been detected in the mussel M. coruscus exposed to OA,17

436

which is highly consistent with our results based on foot transcriptome and RT-qPCR analyses. At the

437

protein level, the abundance analysis illuminated decreased preCOL abundance in byssal threads of P.

438

fucata under the OA condition. Consequently, we reason that the decline in the expression levels of

439

collagen genes in foot tissue may reduce the collagen content in byssal threads, influencing the

440

ultrastructure and morphology of distal threads in the pearl oyster P. fucata.

441

OA can affect marine organisms in multiple ways. Although CaCO3 structures have received the

442

most attention, our mechanistic study reveals that the pearl oyster byssus, a key biological structure in

443

marine ecosystems, is also susceptible to OA stress. In general, OA has negative impacts on

444

production and mechanical properties of distal threads (Figure 6), which will elevate predation and

445

dislodgement risks, leading to increased mortality in the wild pearl oyster population and decreased

446

pearl yield in the cultured population. Therefore, the results of this study provide a foundation for

447

further elucidating the impacts of OA on marine ecosystems as well as shellfish aquaculture from a

448

different perspective.

449 450

SUPPORTING INFORMATION

451

Supplemental experimental details, measured and calculated seawater parameters (Table S1), genes

452

and primers used for RT-qPCR (Table S2), abundance analysis for byssal proteins (Table S3),

453

sequencing and assembly qualities of the mantle transcriptome (Table S4 and Table S5), differentially

454

expressed genes based on transcriptome sequencing (Table S6), SDS-PAGE electrophoresis of byssal

455

proteins (Figure S1), correlation analysis for RT-qPCR and the transcriptome (Figure S2) and



456

enriched pathways (Figure S3- Figure S8). This material is available free of charge via the Internet at

457

http://pubs.acs.org.

458

AUTHOR INFORMATION

459

Corresponding Authors

460

∗

461


462

∗

463


464

Notes

465

The authors declare no competing financial interests.

466

ACKNOWLEDGEMENTS

467

The authors would like to thank all editors and reviewers for their helpful comments on the

468

manuscript. This work was supported by the National Natural Science Foundation of China Grants

469

31502139 and 31572594 and the China Postdoctoral Science Foundation Funded Project Grant

470

2014M550748.

471

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Invert. Surviv. J. 2011, 8 (1), 70-77.

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(48) Wang, X.; Wang, M.; Xu, J.; Jia, Z.; Liu, Z.; Wang, L.; Song, L., Soluble adenylyl cyclase

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mediates mitochondrial pathway of apoptosis and ATP metabolism in oyster Crassostrea gigas

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exposed to elevated CO2. Fish Shellfish Immun. 2017, 66, 140-147.

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(49) Kvitt, H.; Kramarsky-Winter, E.; Maor-Landaw, K.; Zandbank, K.; Kushmaro, A.; Rosenfeld, H.; Fine,

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M.; Tchernov, D., Breakdown of coral colonial form under reduced pH conditions is initiated in polyps and

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mediated through apoptosis. Proc. Nat. Acad. Sci. USA 2015, 112, (7), 2082 -2086.

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(50) Chen, G.; Han, K.; Xu, X.; Du, X.; Zhang, Z.; Tang, J.; Shi, M.; Wang, M.; Li, J.; Cao, B.; Mao,

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X., An anti-leishmanial thiadiazine agent induces multiple myeloma cell apoptosis by suppressing the

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nuclear factor kappa B signalling pathway. Brit. J. Cancer 2014, 110 (1), 63-70.

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(51) Luo, Y.; Li, C.; Landis, A. G.; Wang, G.; Stoeckel, J.; Peatman, E., Transcriptomic profiling of

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differential responses to drought in two freshwater mussel species, the giant floater Pyganodon

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grandis and the Pondhorn Uniomerus tetralasmus. Plos One 2014, 9 (2), e89481.

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(52) Humphrey, J. D.; Dufresne, E. R.; Schwartz, M. A., Mechanotransduction and extracellular

615

matrix homeostasis. Nature 2014, 15 (12), 802-812.



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(53) Andrade, G. R.; Araújo, J. L. F. D.; Nakamura Filho, A.; Guañabens, A. C. P.; Carvalho, M. D. D.;

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Cardoso, A. V. Functional Surface of the golden mussel's foot: morphology, structures and the role of cilia

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on underwater adhesion. Mater. Sci. Eng. C 2015, 54, 32-42.

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(54) Yu, J.; Wei, W.; Danner, E.; Ashley, R. K.; Israelachvili, J. N.; Waite, J. H. Mussel protein adhesion

620

depends on interprotein thiol-mediated redox modulation. Nat. Chem. Biol. 2011, 7 (9), 588-590.

621 622

Figure Legends

623

Figure 1. Effects of ocean acidification on byssus performance in Pinctada fucata. (a) Byssus number,

624

(b) diameter, (c) breaking force and (d) breaking strain of distal threads in the pearl oysters.

625

Figure 2. Effects of ocean acidification on the ultrastructure of distal threads in Pinctada fucata. The

626

ultrastructure at the transverse section of the core in distal threads exposed to the control condition at

627

pH 8.1 (a) and acidified seawater at pH 7.5 (b) were observed by transmission electron microscopy.

628

The yellow circles indicate protein fibers. NC: Nanocavity. Bar = 500 nm.

629

Figure 3. Effects of ocean acidification on the thermal stability of byssal proteins in distal threads of

630

Pinctada fucata. Thermal gravimetric analyses were conducted on distal threads exposed to the

631

control condition at pH 8.1 (a) and acidified seawater at pH 7.5 (b). The rate of weight loss is

632

presented as Deriv. weight (%/°C).

633

Figure 4. Effects of ocean acidification on byssal protein secondary structure in distal threads of

634

Pinctada fucata. Secondary structures were determined by Fourier transform infrared spectroscopy for

635

byssal proteins in distal threads of the pearl oysters exposed to the control condition at pH 8.1 (a) and

636

acidified seawater at pH 7.5 (b). The ratio of secondary structure is presented in each figure.

637

Figure 5. Effects of ocean acidification on the gene expression profile of foot tissue in Pinctada

638

fucata. (a) Scatterplot showing differentially expressed genes (DGEs, p < 0.001 and fold change > 2.0)

639

in foot tissue in Pinctada fucata exposed to the control condition (pH 8.1) and acidified seawater (pH

640

7.5). Color spots indicate the gene expression level: up-regulated genes (red), down-regulated genes

641

(green) and non-DGEs (blue). (b) Fold changes and distributions of DGEs. The digits in the figure

642

indicate the numbers of DGEs between each fold interval.


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643

Figure 6. Schematic diagram for the influencing mechanism of ocean acidification on byssus

644

performance in Pinctada fucata. CO2-driven ocean acidification (OA) alters the abundance and

645

secondary structure of byssal proteins and affects the contents of metal ions in distal threads, which

646

can together reduce the byssus diameter and amplify byssus nanocavity, causing reductions in

647

mechanical properties. Meanwhile, OA alters foot transcriptome, which may influence foot

648

locomotion physiology, leading to a decrease in byssus production.

649

Tables

650

Table 1. Effects of ocean acidification on metal ion contents of distal threads in Pinctada fucata, as

651

detected by inductively coupled plasma mass spectrometry. The asterisks (**) indicate significant

652

differences (p < 0.01) in metal ion contents in byssal threads exposed to acidified seawater (pH 7.5)

653

and the control condition (pH 8.1). Treatment

Metal ion content (mg metal g byssus-1) Ca2+

Si4+

Al3+

Fe3+

Zn2+

pH8.1

19.0 ± 1.25

0.95 ± 0.08

1.20 ± 0.31

0.20 ± 0.06

0.89 ± 0.14

pH7.5

0.07 ± 0.01**

0.40 ± 0.08**

0.60 ± 0.10**

0.35 ± 0.06**

1.31 ± 0.24**

654



Page 26 of 34

655

Table 2. Effects of ocean acidification on byssal protein abundance of distal threads in Pinctada fucata. PSMs: Peptide spectrum matches. vWFA: von

656

Willebrand factor, type A. Asterisks (**, p < 0.01) indicate significant differences in byssal protein abundance in distal threads exposed to acidified

657

seawater (pH 7.5) and the control condition (pH 8.1). Treatment Byssal protein

Fold change in PSMs pH8.1

Matched domain

pH7.5

(Symbol)

(OA/Control) Coverage

PSMs

Coverage

PSMs

TSP1+vWFA

67.51 ± 5.18

268.00 ± 17.88

70.21 ± 15.55

146.25 ± 18.21 **

0.55

vWFA

72.04 ± 19.09

303.00 ± 58.93

65.68 ± 17.77

137.75 ± 15.46 **

0.45

Collagen alpha-4(VI)

64.17 ± 10.65

203.77 ± 13.98

69.03 ± 9.44

24.16 ± 2.10 **

0.12

Peroxidase

56.36 ± 5.37

74.25 ± 10.84

51.03 ± 12.45

144.25 ± 32.18 **

1.94

Thrombospondin 1 containing protein (TSP-1) Foot uncharacterized protein 1 (P-UF1) Collagen (COL) Peroxidase (PER)

658 659


Page 27 of 34


660

Table 3. Enrichment of GO terms of the foot transcriptome of Pinctada fucata exposed to acidified

661

seawater (pH 7.5) compared with the control condition (pH 8.1). FDR-adjusted p < 0.01 indicates

662

significant enrichment. GO term

Category

Gene number

Genes in term

Adjusted p-value

30

2246

4.70e-04

22

2697

7.71e-03

Proton-transporting twoCellular sector ATPase complex component (GO:0016469) Biological

Autophagic cell death

process

(GO:0048102)

663 664

Table 4. Enrichment of KEGG pathways of the foot transcriptome of Pinctada fucata exposed to

665

acidified seawater (pH 7.5) compared with the control condition (pH 8.1). FDR-adjusted p < 0.01

666

indicates significant enrichment. Regulation

Pathway

Gene number

Genes in term

Adjusted p-value

69

773

2.69E-03

144

677

9.65E-07

186

942

1.13E-06

98

429

2.06E-06

113

560

9.48E-05

ECM-receptor interaction Down-regulation (KO04512) Lysosome (KO04514) Ubiquitin mediated proteolysis (KO00061) Up-regulation

Apoptosis (KO04120) NF-kappa β signaling pathway (KO04064)

667 668



Figure 1 102x69mm (300 x 300 DPI)


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Figure 2 60x24mm (300 x 300 DPI)





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TOC 37x17mm (300 x 300 DPI)


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Influencing Mechanism of Ocean Acidification on ... - ACS Publications

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